Alk3/Alk3b and Smad5 Mediate BMP Signaling during Lymphatic Development in Zebrafish

Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.     

Engineered blood and lymphatic capillaries in 3-D VEGF-fibrin-collagen matrices with interstitial flow

In vitro endothelial cell organization into capillaries is a long standing challenge of tissue engineering. We recently showed the utility of low level interstitial flow in guiding the organization of endothelial cells through a 3-D fibrin matrix-containing covalently bound vascular endothelial growth factor (VEGF). Here this synergistic phenomenon was extended to explore the effects of matrix composition on in vitro capillary morphogenesis of human blood versus lymphatic endothelial cells (BECs and LECs). Different mixtures of fibrin and collagen were used in conjunction with constant concentrations of matrix-bound VEGF and slow interstitial flow over 10 days. Interestingly, the BECs and LECs each showed a distinct preference in terms of organization for matrix composition: LECs organized the most extensively in a fibrin-only matrix, while BEC organization was optimized in the compliant collagen-containing matrices. Furthermore, the BECs and LECs produced architecturally different structures; while BECs organized in thick, branched networks containing wide lumen, the LECs were elongated into slender, overlapping networks with fine lumen. These data demonstrate the importance of the 3-D matrix composition in facilitating and coordinating BEC and LEC capillary morphogenesis, which is important for in vitro vascularization of engineered tissues.     

Mechanoinduction of lymph vessel expansion

In the mammalian embryo, few mechanical signals have been identified to influence organ development and function. Here, we report that an increase in the volume of interstitial or extracellular fluid mechanically induces growth of an organ system, that is, the lymphatic vasculature. We first demonstrate that lymph vessel expansion in the developing mouse embryo correlates with a peak in interstitial fluid pressure and lymphatic endothelial cell (LEC) elongation. In 'loss-of-fluid' experiments, we then show that aspiration of interstitial fluid reduces the length of LECs, decreases tyrosine phosphorylation of vascular endothelial growth factor receptor-3 (VEGFR3), and inhibits LEC proliferation. Conversely, in 'gain-of-fluid' experiments, increasing the amount of interstitial fluid elongates the LECs, and increases both VEGFR3 phosphorylation and LEC proliferation. Finally, we provide genetic evidence that β1 integrins are required for the proliferative response of LECs to both fluid accumulation and cell stretching and, therefore, are necessary for lymphatic vessel expansion and fluid drainage. Thus, we propose a new and physiologically relevant mode of VEGFR3 activation, which is based on mechanotransduction and is essential for normal development and fluid homeostasis in a mammalian embryo.     

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.     

TGF-beta1 is a negative regulator of lymphatic regeneration during wound repair

Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -beta1 (TGF-beta1) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-beta1 on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-beta1 expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-beta1 on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-beta1, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-beta1 to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-beta1 inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-beta1 caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-beta1 during wound repair resulted in lymphatic fibrosis and the coexpression of alpha-smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-beta1 expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-beta1 expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-beta1 expression may be useful in promoting more rapid lymphatic regeneration.     

A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic‐specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination‐based ultramicroscopy to entire wholemount‐immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF‐C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development.     

A Tale of Two Models: Mouse and Zebrafish as Complementary Models for Lymphatic Studies

Lymphatic vessels provide essential roles in maintaining fluid homeostasis and lipid absorption. Dysfunctions of the lymphatic vessels lead to debilitating pathological conditions, collectively known as lymphedema. In addition, lymphatic vessels are a critical moderator for the onset and progression of diverse human diseases including metastatic cancer and obesity. Despite their clinical importance, there is no currently effective pharmacological therapy to regulate functions of lymphatic vessels. Recent efforts to manipulate the Vascular Endothelial Growth Factor-C (VEGFC) pathway, which is arguably the most important signaling pathway regulating lymphatic endothelial cells, to alleviate lymphedema yielded largely mixed results, necessitating identification of new targetable signaling pathways for therapeutic intervention for lymphedema. Zebrafish, a relatively new model system to investigate lymphatic biology, appears to be an ideal model to identify novel therapeutic targets for lymphatic biology. In this review, we will provide an overview of our current understanding of the lymphatic vessels in vertebrates, and discuss zebrafish as a promising in vivo model to study lymphatic vessels.     

β1 integrin regulates MMP-10 dependant tubulogenesis in human lymphatic endothelial cells

Lymphatic vessel growth requires extensive remodeling of the extracellular matrix, a process hypothesized to be related to the expression and function of the matrix metalloproteinases. We used a protein based screening strategy to demonstrate increased matrix matalloproteinase-10 expression in human lymphatic endothelial cells undergoing collagen I induced tubulogenesis. Knock-down experiments showed that matrix metalloproteinase-10 regulated lymphatic endothelial cell tubulogenesis. β1 integrin signaling via the ERK/MAPK pathway increased matrix metalloproteinase-10 mRNA and protein expression in human lymphatic endothelial cells. These findings demonstrate a novel mechanism by which β1 integrin regulates matrix metalloproteinase-10 expression during lymphatic vessel remodeling.     

Microcirculation-on-a-Chip: A Microfluidic Platform for Assaying Blood- and Lymphatic-Vessel Permeability

We developed a microfluidic model of microcirculation containing both blood and lymphatic vessels for examining vascular permeability. The designed microfluidic device harbors upper and lower channels that are partly aligned and are separated by a porous membrane, and on this membrane, blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs) were cocultured back-to-back. At cell-cell junctions of both BECs and LECs, claudin-5 and VE-cadherin were detected. The permeability coefficient measured here was lower than the value reported for isolated mammalian venules. Moreover, our results showed that the flow culture established in the device promoted the formation of endothelial cell-cell junctions, and that treatment with histamine, an inflammation-promoting substance, induced changes in the localization of tight and adherens junction-associated proteins and an increase in vascular permeability in the microdevice. These findings indicated that both BECs and LECs appeared to retain their functions in the microfluidic coculture platform. Using this microcirculation device, the vascular damage induced by habu snake venom was successfully assayed, and the assay time was reduced from 24 h to 30 min. This is the first report of a microcirculation model in which BECs and LECs were cocultured. Because the micromodel includes lymphatic vessels in addition to blood vessels, the model can be used to evaluate both vascular permeability and lymphatic return rate.     

Gene expression profile of lymphatic endothelial cells

The lymphatic system was first described at around the same time as the blood circulation centuries ago, but the biological function elucidation of LECs (lymphatic endothelial cells) is far less than that of BVECs (blood vascular endothelial cells). Since the discovery of molecular markers for LECs and exploration of lymphatic role in tumour metastasis, more attention has been given to basic lymphatic research. Approx. 150 known genes were found to be expressed at the mRNA and protein levels by LECs. These molecules play an important role in lymphangiogenesis, signalling, tumour metastasis, immune function and fluid transport. This review provides a brief outline of gene expression profile of LECs and the molecular biological function, which will give the reader a better understanding about the mechanics of lymphatic function and some pathologies related to the lymphatic system such as lymphoedema, and facilitate advanced scientific research into lymphatic biology.     

Laminin‐421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells

Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC‐CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat‐treatment. The chemotactic activity of LEC‐CM was abolished by immunodepletion with anti‐laminin‐1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC‐CM contained laminin‐421. When melanoma C8161 cells were treated with function‐blocking antibodies to integrin α3 or α6, their chemotactic responses to LEC‐CM were markedly reduced. Furthermore, the knock‐down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC‐CM. These data suggest that laminin‐421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.     

Human endothelial cells are chemotactic to endothelial cell growth factor and heparin

The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.     

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.     

Development of the zebrafish lymphatic system requires VEGFC signaling

Lymphangiogenesis results in the formation of a vascular network distinct from arteries and veins that serves to drain interstitial fluid from surrounding tissues and plays a pivotal role in the immune defense of vertebrates as well as in the progression of cancer and other diseases . In mammals, lymph vessels are lined by endothelial cells possibly sprouting from embryonic veins, and their development appears to be critically dependent on the function of PROX1 and VEGFC signaling . The existence of a lymphatic system in teleosts has been a matter of debate for decades. Here we show on the morphological, molecular, and functional levels that zebrafish embryos develop a lymphatic vasculature that serves to retrieve components of the interstitium to the lymph system. We demonstrate the existence of vessels that are molecularly and functionally distinct from blood vessels and show that the development of these vessels depends on Vegfc and VEGFR-3/Flt4 signaling. These findings imply that the molecular components controlling lymphangiogenesis in zebrafish and mammals are conserved and that the zebrafish lymphatic system develops early enough to allow in vivo observations, lineage tracing, and genetic as well as pharmacological screens.     

Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions

Podoplanin is a small, mucin-like membrane glycoprotein highly expressed by lymphatic but not by blood vascular endothelial cells. Although it was shown to be indispensable for the correct formation and function of the lymphatic vasculature, its precise molecular function has remained unknown. In the present study, we identified the mammalian lectin galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Galectin-8 is a tandem-repeat type galectin, which interacts with cell surface glycoproteins, including certain integrins, as well as with extracellular matrix molecules such as fibronectin. Here we show that, similar to podoplanin, galectin-8 is more highly expressed by lymphatic than by blood vascular endothelial cells, and that it promotes lymphatic endothelial cell adhesion as well as haptotactic migration when immobilized onto a surface, while inhibiting the formation of tube-like structures by lymphatic endothelial cells in a collagen matrix when incorporated into the matrix. Importantly, functions of blood vascular endothelial cells, which lack podoplanin expression, are not affected by galectin-8. These data suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding extracellular matrix, most likely in cooperation with other glycoproteins on the surface of lymphatic endothelial cells.     

An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish

Background

Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.

Methods and Results

Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.

Conclusion

Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.     

Mapping the Distinctive Populations of Lymphatic Endothelial Cells in Different Zones of Human Lymph Nodes

The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.     

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen‐age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior–posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis–diffusion–degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen‐age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.