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INTRODUCTION: Botulinum toxin A (BoTA) is a neurotoxin formed by Clostridium botulinum, with a broad medical application spectrum. While the primary effect of BoTA is on the muscles, the effects of BoTA in other systems including the blood vasculature have already been examined, revealing unexpected actions. However, no studies exist to the best of our knowledge regarding the potential effects of BoTA on the lymphatic vascular system, possessing a critical role in health and disease. Isolated human lymphatic endothelial cells (LECs) were cultured in dedicated in vitro culture systems. The analysis including imaging and cell culture approaches as well as molecular biology techniques is performed to examine the LEC alterations occurring upon exposure to different concentrations of BoTA. MATERIALS AND METHODS: Human LECs were cultured and expanded on collagen-coated petri dishes using endothelial basal medium and the commercial product Botox from Allergan as used for all our experiments. Harvested cells were used in various in vitro functional tests to assess the morphologic and functional properties of the BoTA-treated LECs. Gene expression analysis was performed to assess the most important lymphatic system-related genes and pathways. RESULTS: Concentrations of 1, 5 or 10 U of BoTA did not demonstrate a significant effect regarding the proliferation and migration capacity of the LECs versus untreated controls. Interestingly, even the smallest BoTA dose was found to significantly decrease the cord-like-structure formation capacity of the seeded LECs. Gene expression analysis was used to underpin possible molecular alterations, suggesting no significant effect of BoTA in the modification of gene expression versus the starvation medium control. CONCLUSION: LECs appear largely unaffected to BoTA treatment, with an isolated effect on the cord-like-structure formation capacity. Further work needs to assess the effect of BoTA on the smooth-muscle-cell-covered collecting lymphatic vessels and the possible aesthetic implications of such an effect, due to edema formation. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

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Introduction: Idiopathic systemic capillary leak syndrome (SCLS) is a rare disorder characterized by hemoconcentration, hypoproteinemia and edema. Chronic SCLS (cSCLS) presents as intractable edema, distinguishing it from the classic acute form, and only about 10 cases were reported worldwide. Nevertheless, the underlying pathogenesis of both types is obscure. Case presentation: We report a case of a 58-year-old man with chronic edema persisting for 8 years, complicated by unique chylous polyserous effusions and hypotrichosis, which was successfully relieved by treatment with dexamethasone, intravenous immunoglobulin, and thalidomide. Furthermore, a variant c.5594A>G (p.K1865R) in the MYOF gene was identified as a potentially pathogenic mutation through whole-exome genetic sequencing. The proposed mechanism involves its impact on VEGF signaling, leading to increased capillary permeability. Conclusion: Our case illustrates possible lymphatic capillaries involvement in SCLS, which may plays a potential role in immune disorder, and revealed a possible causative genetic mutation of SCLS.

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Fibrosis occurs in many chronic diseases with lymphatic vascular insufficiency (e.g., kidney disease, tumors, and lymphedema). New lymphatic capillary growth can be triggered by fibrosis-related tissue stiffening and soluble factors, but questions remain for how related biomechanical, biophysical, and biochemical cues affect lymphatic vascular growth and function. The current preclinical standard for studying lymphatics is animal modeling, but in vitro and in vivo outcomes often do not align. In vitro models can also be limited in their ability to separate vascular growth and function as individual outcomes, and fibrosis is not traditionally included in model design. Tissue engineering provides an opportunity to address in vitro limitations and mimic microenvironmental features that impact lymphatic vasculature. This review discusses fibrosis-related lymphatic vascular growth and function in disease and the current state of in vitro lymphatic vascular models while highlighting relevant knowledge gaps. Additional insights into the future of in vitro lymphatic vascular models demonstrate how prioritizing fibrosis alongside lymphatics will help capture the complexity and dynamics of lymphatics in disease. Overall, this review aims to emphasize that an advanced understanding of lymphatics within a fibrotic disease-enabled through more accurate preclinical modeling-will significantly impact therapeutic development toward restoring lymphatic vessel growth and function in patients.

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The transcytosis of lipids through enterocytes occurs through the delivery of lipid micelles to the microvilli of enterocytes, consumption of lipid derivates by the apical plasma membrane (PM) and then their delivery to the membrane of the smooth ER attached to the basolateral PM. The SER forms immature chylomicrons (iChMs) in the ER lumen. iChMs are delivered at the Golgi complex (GC) where they are subjected to additional glycosylation resulting in maturation of iChMs. ChMs are secreted into the intercellular space and delivered into the lumen of lymphatic capillaries (LCs). The overloading of enterocytes with lipids induces the formation of lipid droplets inside the lipid bilayer of the ER membranes and transcytosis becomes slower. Here, we examined components of the enterocyte-to-lymphatic barriers in newly born rats before the first feeding and after it. In contrast to adult animals, enterocytes of newborns rats exhibited apical endocytosis and a well-developed subapical endosomal tubular network. These enterocytes uptake membranes from amniotic fluid. Then these membranes are transported across the polarized GC and secreted into the intercellular space. The enterocytes did not contain COPII-coated buds on the granular ER. The endothelium of blood capillaries situated near the enterocytes contained only a few fenestrae. The LCs were similar to those in adult animals. The first feeding induced specific alterations of enterocytes, which were similar to those observed after the lipid overloading of enterocytes in adult rats. Enlarged chylomicrons were stopped at the level of the LAMP2 and Neu1 positive post-Golgi structures, secreted, fused, delivered to the interstitial space, captured by the LCs and transported to the lymph node, inducing the movement of macrophages from lymphatic follicles into its sinuses. The macrophages captured the ChMs, preventing their delivery into the blood.

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CD200 is a cell membrane glycoprotein that interacts with its structurally related receptor (CD200R) expressed on immune cells. We characterized CD200-CD200R interactions in human adult/juvenile (j/a) and fetal (f) skin and in in vivo prevascularized skin substitutes (vascDESS) prepared by co-culturing human dermal microvascular endothelial cells (HDMEC), containing both blood (BEC) and lymphatic (LEC) EC. We detected the highest expression of CD200 on lymphatic capillaries in j/a and f skin as well as in vascDESS in vivo, whereas it was only weakly expressed on blood capillaries. Notably, the highest CD200 levels were detected on LEC with enhanced Podoplanin expression, while reduced expression was observed on Podoplanin-low LEC. Further, qRT-PCR analysis revealed upregulated expression of some chemokines, including CC-chemokine ligand 21 (CCL21) in j/aCD200+ LEC, as compared to j/aCD200- LEC. The expression of CD200R was mainly detected on myeloid cells such as granulocytes, monocytes/macrophages, T cells in human peripheral blood, and human and rat skin. Functional immunoassays demonstrated specific binding of skin-derived CD200+ HDMEC to myeloid CD200R+ cells in vitro. Importantly, we confirmed enhanced CD200-CD200R interaction in vascDESS in vivo. We concluded that the CD200-CD200R axis plays a crucial role in regulating tissue inflammation during skin wound healing.

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CD157 acts as a receptor, regulating leukocyte trafficking and the binding of extracellular matrix components. However, the expression pattern and the role of CD157 in human blood (BEC) and the lymphatic endothelial cells (LEC) of human dermal microvascular cells (HDMEC), remain elusive. We demonstrated constitutive expression of CD157 on BEC and LEC, in fetal and juvenile/adult skin, in situ, as well as in isolated HDMEC. Interestingly, CD157 epitopes were mostly localized on BEC, co-expressing high levels of CD31 (CD31High), as compared to CD31Low BEC, whereas the podoplanin expression level on LEC did not affect CD157. Cultured HDMEC exhibited significantly higher numbers of CD157-positive LEC, as compared to BEC. Interestingly, separated CD157- and CD157+ HDMEC demonstrated no significant differences in clonal expansion in vitro, but they showed distinct expression levels of cell adhesion molecules, before and after cytokine stimulation in vitro. In particular, we proved the enhanced and specific adherence of CD11b-expressing human blood myeloid cells to CD157+ HDMEC fraction, using an in vitro immune-binding assay. Indeed, CD157 was also involved in chemotaxis and adhesion of CD11b/c monocytes/neutrophils in prevascularized dermo-epidermal skin substitutes (vascDESS) in vivo. Thus, our data attribute specific roles to endothelial CD157, in the regulation of innate immunity during inflammation.

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Pancreatic cancer is associated with a poor prognosis, mainly due to lymph node invasion and lymph node recurrence after surgical resection, even after extended lymphadenectomy. The peripancreatic lymphatic system is highly complex and the specific lymphatic drainage of each part of the pancreas has not been established. The aim of this study was to determine the lymphatic drainage pathways specific to each part of the pancreas on live pigs using Patent Blue. The pancreases of 14 live pigs were injected in different parts of the gland. The technique was efficient and reproducible. The diffusion patterns were similar for each location and were reported. Our results in pigs allowed us to define specific nodal relay stations and lymphatic drainage for each part of the pancreas and confirm that independent anatomical-surgical pancreatic segments can be described. It is interesting to note that lymphatic drainage for the upper part of the proximal part of pancreas (duodenal lobe) occurred on the left side of the portal vein. This suggests that lymph node resection during cephalic duodenopancreatectomy in humans should be extended to the left side of the mesenteric vein, and probably to the right side of the superior mesenteric artery, as recently suggested. These results could help surgeons perform safe anatomical-segmental pancreatic resections with accurate lymphadenectomies and improve survival in patients with pancreatic cancer. Based on these results we will perform an innovative prospective study. Patent Blue will be injected into different parts of the gland in patients operated for pancreatic resection, and lymphatic diffusion of the dye will be recorded in relation to their origin from the theoretical pancreatic segments (ClinicalTrials.gov Identifier: NCT03597230).

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An endothelial cell monolayer separates interstitia from blood and lymph, and determines the bidirectional transfer of solutes and macromolecules across these biological spaces. We review advances in transport modalities across these endothelial barriers. Glucose is a major fuel for the brain and peripheral tissues, and insulin acts on both central and peripheral tissues to promote whole-body metabolic signalling and anabolic activity. Blood-brain barrier endothelial cells display stringent tight junctions and lack pinocytic activity. Delivery of blood glucose and insulin to the brain occurs through their respective carrier (Glucose transporter 1) and receptor (insulin receptor), enacting bona fide transcytosis. At supraphysiological concentrations, insulin is also likely transferred by fluid phase cellular uptake and paracellular transport, especially in peripheral microvascular endothelia. The lymphatic microvasculature also transports insulin but in this case from tissues to lymph and therefrom to blood. This serves to end the hormone's action and to absorb highly concentrated subcutaneously injected insulin in diabetic individuals. The former function may involve receptor-mediated transcytosis into lymphatic endothelial cells, the latter fluid phase uptake and paracellular transport. Lymphatic capillaries also mediate carrier-dependent transport of other nutrients and macromolecules. These findings challenge the notion that lymphatic capillaries only transport macromolecules through intercellular flaps.

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Surgical laparoscopic procedures in the retroperitoneal and supramesocolic spaces are increasingly frequent. There is a high risk of iatrogenic intraoperative injury of the retroperitoneal lymphatic structures during these procedures. A precise understanding of the anatomy of the thoracic duct (TD) and the cisterna chyli (CC) is essential for safe surgical procedures in this area. However, routine imaging procedures rarely and often incorrectly visualize the CC. The objective of this study was to evaluate the feasibility of a retrograde injection of the TD to fill the CC with a contrast agent in 16 human cadavers. Both magnetic resonance lymphography (MRI) and computed tomography (CT) studies could be performed on the same anatomical specimen, using a contrast medium which hardened, allowing gross dissection. MRI and CT detectability were evaluated, and imaging results were compared with the anatomical dissection. The CC of 12/16 cadavers were successfully injected, and four were unsuccessful due to technical difficulties, showing the effectiveness of the method. This technique can improve understanding of the anatomy of the TD and CC and provides an original option to study the complex anatomy of these structures by correlating precise cadaveric dissections with cross-sectional imaging.

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The lymphatic vasculature plays an essential role in the maintenance of tissue interstitial fluid balance and in the immune response. After capture of fluids, proteins and antigens by lymphatic capillaries, lymphatic collecting vessels ensure lymph transport. An important component to avoid lymph backflow and to allow a unidirectional flow is the presence of intraluminal valves. Defects in the function of collecting vessels lead to lymphedema. Several important factors and signaling pathways involved in lymphatic collecting vessel maturation and valve morphogenesis have now been discovered. The present review summarizes the current knowledge about the key steps of lymphatic collecting vessel development and maturation and focuses on the regulatory mechanisms involved in lymphatic valve formation.

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Lymphatic vasculature is necessary for maintaining fluid homeostasis in vertebrates. During embryogenesis lymphatic endothelial cells originate from the veins as a homogeneous population. These cells undergo a series of changes at the morphological and molecular levels to become mature lymphatic vasculature that consists of lymphatic capillaries, collecting lymphatic vessels and valves. In this article we summarize our current knowledge about these steps and highlight some black boxes that require further clarification.

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Any method that deforms the skin of the extremities may increase lymphatic flow rate, and may be applied to treat peripheral lymphedema. This study was undertaken to investigate whether or not elastic adhesive tape with passive exercise can increase lymph flow in the rabbit hind leg by effective and periodic skin deformation. Cannulation into a pre-popliteal afferent lymphatic vessel in the lower left leg of 22 male New Zealand White rabbits was performed under a stereomicroscope. After stabilization, lymph was collected at rest or during passive exercise with an electric motor at 60 r.p.m. for 15 minutes and was then measured. Lymph flow rate was calculated and expressed as g/hour. Increase of lymph flow rate due to taping was significant only for passive exercise (p=0.0317). The lymph flow rate increased linearly as the area of tape was increased (p=0.0011), and lymph flow rates were significantly different according to site (p=0.0017). Tape on the anterior aspect of the ankle caused salient deformation and tended to increase the lymph flow rate more so than tape on the dorsum of the foot (p=0.0831). Taping with elastic adhesive tape in passive exercise increased the lymph flow rate in the rabbit hind leg by deforming the skin, which suggests a novel therapeutic method in cases of peripheral lymphedema.

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