Prox1, master regulator of the lymphatic vasculature phenotype

In contrast to the extensive molecular and functional characterization of blood vascular endothelium, little is known about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels. The homeobox gene Prox1, the vertebrate homologue of the Drosophila prospero gene, has been recently identified to be required for the induction of lymphatic vascular development from preexisting embryonic veins, and studies in Prox1-deficient mice have confirmed Florence Sabin's original hypothesis about the origin of the lymphatic vascular system from embryonic veins. The recent establishment of cell culture models for the selective propagation of blood vascular and lymphatic endothelial cells, together with the findings that these cells maintain their lineage-specific differentiation in vitro, has led to the discovery that Prox1 expression is sufficient to induce a lymphatic phenotype in blood vascular endothelium. Ectopic expression of Prox1 downregulated blood vascular-associated genes and also upregulated some of the known lymphatic endothelial cell markers. Together, these studies suggest that the blood vascular phenotype represents the default endothelial differentiation and they identify an essential role of Prox1 in the program specifying lymphatic endothelial cell fate.     

Segmental territories along the cardinal veins generate lymph sacs via a ballooning mechanism during embryonic lymphangiogenesis in mice

During lymphangiogenesis in the mammalian embryo, a subset of vascular endothelial cells in the cardinal veins is reprogrammed to adopt a lymphatic endothelial fate. The prevailing model of lymphangiogenesis contends that these lymphatic precursor cells migrate away from the cardinal veins and reassemble peripherally as lymph sacs from which a lymphatic vasculature is generated. However, this model fails to account for a number of observations that, as a result, have remained anecdotal. Here, we use optical projection tomography, confocal microscopy and in vivo live imaging to uncover three key stages of lymphatic vascular morphogenesis in the mouse embryo at high resolution. First, we define territories or "pre-lymphatic clusters" of Prox1-positive lymphatic endothelial progenitor cells along the antero-posterior axis of the cardinal veins. Second, these pre-lymphatic clusters undergo progressive extrusion ("ballooning") to generate primitive lymph sacs. Third, lymphatic vessels emerge by a combination of mechanisms including sprouting from the lymph sacs and direct delamination of streams of cells from the cardinal veins. Our data support a new model for lymphatic vascular patterning and morphogenesis, as a basis for identifying the molecular cues governing these processes.     

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.     

Biomarkers of lymphatic function and disease: state of the art and future directions

Substantial advances have accrued over the last decade in the identification of the processes that contribute to lymphatic vascular development in health and disease. Identification of distinct regulatory milestones, from a variety of genetic models, has led to a stepwise chronology of lymphatic development. Several molecular species have been identified as important tissue biomarkers of lymphatic development and function. At present, vascular endothelial growth-factor receptor (VEGFR)-3/VEGF-C/VEGF-D signaling has proven useful in the identification of clinical lymphatic metastatic potential and the assessment of cancer prognosis. Similar biomarkers, to be utilized as surrogates for the assessment of inherited and acquired diseases of the lymphatic circulation, are actively sought, and will represent a signal advance in biomedical investigation.     

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.     

Generation of non-permissive basement membranes by anti-laminin antibody fragments produced by matrix-embedded gene-modified cells

Tumor-induced blood vessel formation is a key process for the growth and spread of solid tumors, traditionally attributed to activated host endothelial cells (angiogenesis). Recently, highly aggressive cancer cells have been shown to form vascular channels in the absence of endothelial cells (vasculogenic mimicry). In this work, we have focused on the common dependence of both processes in their interactions with the surrounding extracellular matrix. We had previously described a human recombinant anti-laminin antibody that blocked the capillary morphogenesis of human endothelial cells. Here, we demonstrate that the purified antibody is capable of inhibiting channel formation by human cancer cells, suggesting a common morphogenic pathway in both processes. Moreover, matrix-embedded cells producing antibody fragments may render the surrounding matrix non-permissive for aggressive tumor cells. These results open the way for the development of new therapeutic strategies for cancer.     

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.     

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.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype

The process of angiogenesis has been well documented, but little is known about the biology of lymphatic endothelial cells and the molecular mechanisms controlling lymphangiogenesis. The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. In Prox1(-)(/-) embryos, this budding becomes arrested at around embryonic day (E)11.5, resulting in embryos without lymphatic vasculature. Unlike the endothelial cells that bud off in E11.5 wild-type embryos, those of Prox1-null embryos did not co-express any lymphatic markers such as VEGFR-3, LYVE-1 or SLC. Instead, the mutant cells appeared to have a blood vascular phenotype, as determined by their expression of laminin and CD34. These results suggest that Prox1 activity is required for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype. On the basis of our findings, we propose that a blood vascular phenotype is the default fate of budding embryonic venous endothelial cells; upon expression of Prox1, these budding cells adopt a lymphatic vasculature phenotype.     

Hepatocyte growth factor promotes lymphatic vessel formation and function

The lymphatic vascular system plays a pivotal role in mediating tissue fluid homeostasis and cancer metastasis, but the molecular mechanisms that regulate its formation and function remain poorly characterized. A comparative analysis of the gene expression of purified lymphatic endothelial cells (LEC) versus blood vascular endothelial cells (BVEC) revealed that LEC express significantly higher levels of hepatocyte growth factor receptor (HGF-R). Whereas little or no HGF-R expression was detected by lymphatic vessels of normal tissues, HGF-R was strongly expressed by regenerating lymphatic endothelium during tissue repair and by activated lymphatic vessels in inflamed skin. Treatment of cultured LEC with HGF promoted LEC proliferation, migration and tube formation. HGF-induced proliferation of LEC did not require vascular endothelial growth factor receptor-3 activation, and HGF-induced cell migration was partially mediated via integrin alpha-9. Transgenic or subcutaneous delivery of HGF promoted lymphatic vessel formation in mice, whereas systemic blockade of HGF-R inhibited lymphatic function. These results identify HGF as a novel, potent lymphangiogenesis factor, and also indicate that HGF-R might serve as a new target for inhibiting pathological lymphangiogenesis.     

Development of a new method for isolation and long-term culture of organ-specific blood vascular and lymphatic endothelial cells of the mouse

Endothelial cells are indispensable components of the vascular system, and play pivotal roles during development and in health and disease. Their properties have been studied extensively by in vivo analysis of genetically modified mice. However, further analysis of the molecular and cellular phenotypes of endothelial cells and their heterogeneity at various developmental stages, in vascular beds and in various organs has often been hampered by difficulties in culturing mouse endothelial cells. In order to overcome these difficulties, we developed a new transgenic mouse line expressing the SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary expression system based on Cre/loxP recombination. tsA58T Ag-positive endothelial cells in primary cultures of a variety of organs proliferate continuously at 33 degrees C without undergoing cell senescence. The resulting cell population consists of blood vascular and lymphatic endothelial cells, which could be separated by immunosorting. Even when cultured for two months, the cells maintained endothelial cell properties, as assessed by expression of endothelium-specific markers and intracellular signaling through the vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3, as well as their physiological characteristics. In addition, lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an organ-specific endothelial characteristic was maintained. These results show that our transgenic cell culture system is useful for culturing murine endothelial cells, and will provide an accessible method and applications for studying endothelial cell biology.     

In Vitro Angiogenic and Proteolytic Properties of Bovine Lymphatic Endothelial Cells

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-l, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.     

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.M. Matsuo and K. Koizumi contributed equally to this work. This study was supported in part by Grants-in-Aid for the 21st Century COE Program and for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo

The lymphatic vascular system functions to maintain fluid homeostasis by removing fluid from the interstitial space and returning it to venous circulation. This process is dependent upon the maintenance and modulation of a semi-permeable barrier between lymphatic endothelial cells of the lymphatic capillaries. However, our understanding of the lymphatic endothelial barrier and the molecular mechanisms that govern its function remains limited. Adrenomedullin (AM) is a 52 amino acid secreted peptide which has a wide range of effects on cardiovascular physiology and is required for the normal development of the lymphatic vascular system. Here, we report that AM can also modulate lymphatic permeability in cultured dermal microlymphatic endothelial cells (HMVEC-dLy). AM stimulation caused a reorganization of the tight junction protein ZO-1 and the adherens protein VE-cadherin at the plasma membrane, effectively tightening the endothelial barrier. Stabilization of the lymphatic endothelial barrier by AM occurred independently of changes in junctional protein gene expression and AM^−/− endothelial cells showed no differences in the gene expression of junctional proteins compared to wildtype endothelial cells. Nevertheless, local administration of AM in the mouse tail decreased the rate of lymph uptake from the interstitial space into the lymphatic capillaries. Together, these data reveal a previously unrecognized role for AM in controlling lymphatic endothelial permeability and lymphatic flow through reorganization of junctional proteins.     

VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism

BACKGROUND: The lymphatic endothelium is an important semi-permeable barrier separating lymph from the interstitial space. However, there is currently a limited understanding of the lymphatic endothelial barrier and the mechanisms of lymph formation. The objectives of this study were to investigate the potential active role of lymphatic endothelial cells in barrier regulation, and to test whether the endothelial cell agonists VEGF-A and VEGF-C can alter lymphatic endothelial barrier function. METHODS AND RESULTS: Cultured adult human dermal microlymphatic endothelial cells (HMLEC-d) and human umbilical vein endothelial cells (HUVEC) were respectively used as models of lymphatic and vascular endothelium. Transendothelial electrical resistance (TER) of endothelial monolayers served as an index of barrier function. Cells were treated with VEGF-A, VEGF-C, or the VEGFR-3 selective mutant VEGF-C156S. MAZ51 was used to inhibit VEGFR-3 signaling. The results show that while VEGF-A causes a time-dependent decrease in TER in HUVEC, there is no response in HMLEC-d. In contrast, VEGF-C and VEGF-C156S cause a similar decrease in TER in HMLEC-d that is not observed in HUVEC. These results corresponded to the protein expression of VEGFR-2 and VEGFR-3 in these cell types, determined by Western blotting. In addition, the VEGF-C- and VEGF-C156S-induced TER changes were inhibited by MAZ51. CONCLUSIONS: The results indicate differential responses of the lymphatic and vascular endothelial barriers to VEGF-A and VEGF-C. Furthermore, our data suggest that VEGF-C alters lymphatic endothelial function through a mechanism involving VEGFR-3.     

Immunohistochemical demonstration of Angiopoietin-2 in lymphatic vascular development

The cellular expression of Angiopoietin-2 (Ang2) was studied during lymphatic development in mouse by immunohistochemistry and compared to that of lymphatic endothelial markers. At the earliest stage of lymphvasculogenesis, Prox1-identified lymphatic precursor cells of the cardinal vein displayed an intense immunoreaction for Ang2 in their cytoplasm, implying that Ang2 may adjust lymphatic specification and sprouting from the veins under the control of Prox1. Thereafter, Ang2 was constantly expressed in Prox1 and/or LYVE-1-immunopositive endothelial cells of lymphatic sacs and vessels, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development, and the lymphatic endothelial cells simultaneously exhibited immunoreactivity to Tie2, a primary receptor for angiopoietins. These results suggest that lymphatic endothelial cells may regulate lymphatic development via their own Ang2-Tie2 signaling. Ang2 is further immunolocalized in the developing blood vessels including hepatic sinusoids, adrenal medullary vasculature and postnatal pulmonary vessels, thereby indicating that the blood vessels, which undergo vascular remodeling and sudden alteration of blood flow during the development, are also likely to express Ang2. The present study is first to demonstrate Ang2 expression in the lymphatic endothelial cells during development, and consequently Ang2 is regarded as a molecular profile of the developing lymphatic endothelial cells required for lymphatic vascular organization.     

Anticancer properties for 4,4'-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007)/3,7-diaminophenothiazin-5-ium double salts

4,4'-Dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007) formed stable double salts with phenothiazin-5-ium salts (2a-d), which have improved in vitro anticancer activities, as compared to A-007 alone. The stable salt between methylene blue (2a) and A-007 allowed the latter to diffuse into the dermis layers of skin. It is anticipated that these new salts will allow A-007 to penetrate into the deep lymphatic/vascular channels of the dermis, which contain metastatic cancer cells, and improve in vivo anticancer activities.