LTßR Signaling Controls Lymphatic Migration of Immune Cells.

The pleiotropic functions of lymphotoxin (LT)ß receptor (LTßR) signaling are linked to the control of secondary lymphoid organ development and structural maintenance, inflammatory or autoimmune disorders, and carcinogenesis. Recently, LTßR signaling in endothelial cells has been revealed to regulate immune cell migration. Signaling through LTßR is comprised of both the canonical and non-canonical-nuclear factor κB (NF-κB) pathways, which induce chemokines, cytokines, and cell adhesion molecules. Here, we focus on the novel functions of LTßR signaling in lymphatic endothelial cells for migration of regulatory T cells (Tregs), and specific targeting of LTßR signaling for potential therapeutics in transplantation and cancer patient survival.

A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutation in these isolated cells.

PURPOSE: Tissue disaggregation and the cell sorting technique by surface markers has played an important role in isolating lymphatic endothelial cells (LECs) from lymphatic malformation (LM). However, this technique may have the drawback of impurities or result in isolation failure because it is dependent on surface marker expressions, the heterogeneity of which has been found in the lymphatic system. We developed a novel method for isolating LM-LECs without using whole tissue disaggregation. METHODS: Seven LM surgical specimens were collected from seven patients with LMs. LM-LECs were detached from the LM cyst wall by "lumen digestion" and irrigating the cystic cavity with trypsin, and maintained in culture. RESULTS: The cells formed a monolayer with a cobblestone-like appearance. Immunohistochemistry and quantitative RT-PCR of these cells revealed high expression of lymphatic-specific genes, confirming their identity as LM-LECs. The whole-exome sequencing and PIK3CA sequencing of these cells revealed somatic mutations in PIK3CA in all cases. CONCLUSIONS: We established a novel technique for isolating LM-LECs from LM tissue by "lumen digestion" without whole-tissue disaggregation. The limited incorporation of non-LM LECs in the isolate in our method could make it an important tool for investigating the heterogeneity of gene expression as well as mutations in LM-LECs.

Pelvic sentinel lymph node biopsy in endometrial cancer-a simplified algorithm based on histology and lymphatic anatomy.

OBJECTIVE: To achieve the full potential of sentinel lymph node (SLN) detection in endometrial cancer, both presumed low- and high-risk groups should be included. Perioperative resource use and complications should be minimized. Knowledge on distribution and common anatomical sites for metastatic SLNs may contribute to optimizing the concept while maintaining sensitivity. Proceeding from previous studies, simplified algorithms based on histology and lymphatic anatomy are proposed. METHODS: Data on mapping rates and locations of pelvic SLNs (metastatic and non-metastatic) from two previous prospective SLN studies in women with endometrial cancer were retrieved. Cervically injected indocyanine green was used as a tracer and an ipsilateral re-injection was performed in case of non-display of the upper and/or lower paracervical pathways. A systematic surgical algorithm was followed with clearly defined SLNs depicted on an anatomical chart. In high-risk endometrial cancer patients, removal of SLNs was followed by a pelvic and para-aortic lymphadenectomy. RESULTS: 423 study records were analyzed. The bilateral mapping rates of the upper and lower paracervical pathways were 88.9% and 39.7%, respectively. 72% of all SLNs were typically positioned along the upper paracervical pathway (interiliac and/or proximal obturator fossa) and 71 of 75 (94.6%) of pelvic node positive women had at least one metastatic SLN at either of these positions. Women with grade 1-2 endometroid cancers (n=275) had no isolated metastases along the lower paracervical pathway compared with two women with high-risk histologies (n=148). CONCLUSION: SLNs along the upper paracervical pathway should be identified in all endometrial cancer histological subtypes; removal of nodes at defined typical positions along the upper paracervical pathway may replace a site-specific lymphadenectomy in case of non-mapping despite tracer re-injection. Detection of SLNs along the lower paracervical pathway can be restricted to high-risk histologies and a full pre-sacral lymphadenectomy should be performed in case of non-display.

Non-canonical WNT-signaling controls differentiation of lymphatics and extension lymphangiogenesis via RAC and JNK signaling.

Development of lymphatics takes place during embryogenesis, wound healing, inflammation, and cancer. We previously showed that Wnt5a is an essential regulator of lymphatic development in the dermis of mice, however, the mechanisms of action remained unclear. Here, whole-mount immunostaining shows that embryonic day (ED) 18.5 Wnt5a-null mice possess non-functional, cyst-like and often blood-filled lymphatics, in contrast to slender, interconnected lymphatic networks of Wnt5a+/- and wild-type (wt) mice. We then compared lymphatic endothelial cell (LEC) proliferation during ED 12.5, 14.5, 16.5 and 18.5 between Wnt5a-/-, Wnt5a+/- and wt-mice. We did not observe any differences, clearly showing that Wnt5a acts independently of proliferation. Transmission electron microscopy revealed multiple defects of LECs in Wnt5a-null mice, such as malformed inter-endothelial junctions, ruffled cell membrane, intra-luminal bulging of nuclei and cytoplasmic processes. Application of WNT5A protein to ex vivo cultures of dorsal thoracic dermis from ED 15.5 Wnt5a-null mice induced flow-independent development of slender, elongated lymphatic networks after 2 days, in contrast to controls showing an immature lymphatic plexus. Reversely, the application of the WNT-secretion inhibitor LGK974 on ED 15.5 wt-mouse dermis significantly prevented lymphatic network elongation. Correspondingly, tube formation assays with human dermal LECs in vitro revealed increased tube length after WNT5A application. To study the intracellular signaling of WNT5A we used LEC scratch assays. Thereby, inhibition of autocrine WNTs suppressed horizontal migration, whereas application of WNT5A to inhibitor-treated LECs promoted migration. Inhibition of the RHO-GTPase RAC, or the c-Jun N-terminal kinase JNK significantly reduced migration, whereas inhibitors of the protein kinase ROCK did not. WNT5A induced transient phosphorylation of JNK in LECs, which could be inhibited by RAC- and JNK-inhibitors. Our data show that WNT5A induces formation of elongated lymphatic networks through proliferation-independent WNT-signaling via RAC and JNK. Non-canonical WNT-signaling is a major mechanism of extension lymphangiogenesis, and also controls differentiation of lymphatics.

Lymphatic transport of exosomes as a rapid route of information dissemination to the lymph node.

It is well documented that cells secrete exosomes, which can transfer biomolecules that impact recipient cells' functionality in a variety of physiologic and disease processes. The role of lymphatic drainage and transport of exosomes is as yet unknown, although the lymphatics play critical roles in immunity and exosomes are in the ideal size-range for lymphatic transport. Through in vivo near-infrared (NIR) imaging we have shown that exosomes are rapidly transported within minutes from the periphery to the lymph node by lymphatics. Using an in vitro model of lymphatic uptake, we have shown that lymphatic endothelial cells actively enhanced lymphatic uptake and transport of exosomes to the luminal side of the vessel. Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow. Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake. Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

Neutrophil heterogeneity: implications for homeostasis and pathogenesis.

Neutrophils are polymorphonuclear leukocytes of the phagocytic system that act as first line of host defense against invading pathogens but are also important mediators of inflammation-induced injury. In contrast to other members of the innate immune system, neutrophils are classically considered a homogenous population of terminally differentiated cells with a well-defined and highly conserved function. Indeed, their short lifespan, the absent proliferative capacity, their limited ability to produce large amounts of cytokines, and the failure to recirculate from the tissue to the bloodstream have sustained this idea. However, increasing evidence over the last decade has demonstrated an unexpected phenotypic heterogeneity and functional versatility of the neutrophil population. Far beyond their antimicrobial functions, neutrophils are emerging as decision-shapers during innate and adaptive immune responses. These emerging discoveries open a new door to understand the role of neutrophils during homeostatic but also pathogenic immune processes. Thus, this review details novel insights of neutrophil phenotypic and functional heterogeneity during homeostasis and disease.

Sphingosine-1-phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

Lymphatic and nerve distribution throughout the parametrium.

BACKGROUND: Our objective was to concomitantly assess distribution of lymphatic and nerve structures in the parametrium. METHODS: Twenty hemipelvises from ten fresh cadavers were dissected to differentiate between, three different parts of the parametrium: the lateral parametrium, the proximal and the distal part of the posterior parametrium. Histologic and immunofluorescence analyses of nerve and lymphatic structures were performed using NSE and LYVE-1 staining, respectively. The percentage of structures was independently scored as 0 (0%), 1 (1-20%), 2 (20-50%), 3 (50-80%), 4 (>80%). RESULTS: The lateral parametrium and the proximal part of the posterior parametrium contained both nerve (scored 2.25 and 2.50, respectively) and lymphatic (scored 2.50 and 2.00, respectively) structures. The distal part of the posterior parametrium also contained numerous nerve structures (scored 2.00) but lymphatic structures were rare (scored 0.88). No difference in nerve distribution was found according to the parts of parametrium while a significantly lower distribution of lymphatic vessels was observed in the distal part of the posterior parametrium (p=0.03). CONCLUSION: The distal part of the posterior parametrium is of high nerve density and low lymphatic density raising the issue as to whether it should be removed during radical hysterectomy.

Tissue engineering and regeneration of lymphatic structures.

Tissue engineering is the process by which biological structures are recreated using a combination of molecular signals, cellular components and scaffolds. Although the perceived potential of this approach to reconstruct damaged or missing tissues is seemingly limitless, application of these ideas in vivo has been more difficult than expected. However, despite these obstacles, important advancements have been reported for a number of organ systems, including recent reports on the lymphatic system. These advancements are important since the lymphatic system plays a central role in immune responses, regulation of inflammation, lipid absorption and interstitial fluid homeostasis. Insights obtained over the past two decades have advanced our understanding of the molecular and cellular mechanisms that govern lymphatic development and function. Utilizing this knowledge has led to important advancements in lymphatic tissue engineering, which is the topic of this review.

Near-infrared fluorescent silica-coated gold nanoparticle clusters for x-ray computed tomography/optical dual modal imaging of the lymphatic system.

Lymph nodes (LNs) are often removed to prevent the spread of cancer because they are frequently the first site of metastases. However, the enucleation of LNs requires difficult operative techniques and lymphedema can result as a complication. Although lymphedema can be cured by anastomosis of a lymph vessel (LV) to a vein, the operative procedure is extremely difficult because LNs and LVs are too small and indistinct to be identified. Therefore, visualization of LNs and LVs is important. The combination of X-ray computed tomography (CT) and fluorescence imaging, CT/fluorescence dual modal imaging, enables the visualization of LNs and LVs before and during surgery. To accomplish this, near-infrared fluorescent silica-coated gold nanoparticle clusters (Au@SiO2) with a high X-ray absorption coefficient are synthesized. Both fluorescence imaging and CT show that the Au@SiO2 nanoparticles gradually accumulate in LNs through LVs. CT determines the location and size of the LNs and LVs without dissection, and fluorescence imaging facilitates their identification. The Au@SiO2 nanoparticles have neither hepatotoxicity nor nephrotoxicity. The results demonstrate that CT/fluorescence dual modal imaging using Au@SiO2 nanoparticles provides anatomical information, including the location and size of LNs and LVs for determining a surgery plan, and provides intraoperative visualization of LNs and LVs to facilitate the operation.

The new era of the lymphatic system: no longer secondary to the blood vascular system.

The blood and lymphatic systems are the two major circulatory systems in our body. Although the blood system has been studied extensively, the lymphatic system has received much less scientific and medical attention because of its elusive morphology and mysterious pathophysiology. However, a series of landmark discoveries made in the past decade has begun to change the previous misconception of the lymphatic system to be secondary to the more essential blood vascular system. In this article, we review the current understanding of the development and pathology of the lymphatic system. We hope to convince readers that the lymphatic system is no less essential than the blood circulatory system for human health and well-being.

New model of macrophage acquisition of the lymphatic endothelial phenotype.

BACKGROUND: Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions. METHODOLOGY/PRINCIPAL FINDINGS: Time-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b(+)/VEGFR-3(+) LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels. CONCLUSIONS/SIGNIFICANCE: We present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the "RAW model" mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology.

Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules.

While patrolling the body in search of foreign antigens, naive lymphocytes continuously circulate from the blood, through the lymph nodes, into the lymphatic vessels and back to the blood. This process, called lymphocyte recirculation, provides the body with effective immune surveillance for foreign invaders and for alterations to the body's own cells. However, the mechanisms that regulate lymphocyte recirculation during homeostasis remain incompletely characterized. Here we show that dendritic cells (DCs), which are well known for their role in antigen presentation to T lymphocytes, control the entry of naive lymphocytes to lymph nodes by modulating the phenotype of high endothelial venules (HEVs), which are blood vessels specialized in lymphocyte recruitment. We found that in vivo depletion of CD11c(+) DCs in adult mice over a 1-week period induces a reduction in the size and cellularity of the peripheral and mucosal lymph nodes. In the absence of DCs, the mature adult HEV phenotype reverts to an immature neonatal phenotype, and HEV-mediated lymphocyte recruitment to lymph nodes is inhibited. Co-culture experiments showed that the effect of DCs on HEV endothelial cells is direct and requires lymphotoxin-ß-receptor-dependent signalling. DCs express lymphotoxin, and DC-derived lymphotoxin is important for lymphocyte homing to lymph nodes in vivo. Together, our results reveal a previously unsuspected role for DCs in the regulation of lymphocyte recirculation during immune surveillance.

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.

Intestinal inflammation induces genotoxicity to extraintestinal tissues and cell types in mice.

Chronic intestinal inflammation leads to increased risk of colorectal and small intestinal cancers and is also associated with extraintestinal manifestations such as lymphomas, other solid cancers and autoimmune disorders. We have previously found that acute and chronic intestinal inflammation causes DNA damage to circulating peripheral leukocytes, manifesting a systemic effect in genetically and chemically induced models of intestinal inflammation. Our study addresses the scope of tissue targets and genotoxic damage induced by inflammation-associated genotoxicity. Using several experimental models of intestinal inflammation, we analyzed various types of DNA damage in leukocyte subpopulations of the blood, spleen, mesenteric and peripheral lymph nodes and in intestinal epithelial cells, hepatocytes and the brain. Genotoxicity in the form of DNA single- and double-stranded breaks accompanied by oxidative base damage was found in leukocyte subpopulations of the blood, diverse lymphoid organs, intestinal epithelial cells and hepatocytes. The brain did not demonstrate significant levels of DNA double-stranded breaks as measured by γ-H2AX immunostaining. CD4(+) and CD8(+) T-cells were most sensitive to DNA damage versus other cell types in the peripheral blood. In vivo measurements and in vitro modeling suggested that genotoxicity was induced by increased levels of systemically circulating proinflammatory cytokines. Moreover, genotoxicity involved increased damage rather than reduced repair, as it is not associated with decreased expression of the DNA double-strand break recognition and repair protein, ataxia telangiectasia mutated. These findings suggest that levels of intestinal inflammation contribute to the remote tissue burden of genotoxicity, with potential effects on nonintestinal diseases and cancer.

Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the Drosophila lymph gland.

The Drosophila melanogaster hematopoietic organ, called lymph gland, proliferates and differentiates throughout the larval period. The lymph gland of the late larva is comprised of a large primary lobe and several smaller secondary lobes. Differentiation into two types of hemocytes, plasmatocytes and crystal cells, is confined to the outer layer (cortical zone) of the primary lobe; the center of the primary lobe (medullary zone), as well as the secondary lobes, contain only proliferating prohemocytes. A small cluster of cells located at the posterior tip of the primary lobe serves as a signaling center (PSC) that inhibits precocious differentiation of the medullary zone. The larval lymph gland is stabilized by layers of extracellular matrix (basement membranes) that surround individual hemocytes, groups of hemocytes, as well as the lymph gland as a whole. In this paper, we investigated the events shaping the lymph gland in the early pupa. The lymph gland dissociates and hemocytes disperse during the first 12 h after puparium formation (APF), leaving behind empty husks of basement membrane. Prior to lymph gland dissociation, cells of the medullary zone differentiate, expressing the early differentiation marker Peroxidasin (Pxn), as well as, in part, the late differentiation marker P1. Cells of the PSC spread throughout the pupal lymph gland prior to their dispersal. Cells of the secondary lobes undergo a rapid phase of proliferation that lasts until 8 h APF, followed by expression of Pxn and dispersal. These hemocytes do not express P1, indicating that they disperse prior to full maturation.

miR-31 functions as a negative regulator of lymphatic vascular lineage-specific differentiation in vitro and vascular development in vivo.

The lymphatic vascular system maintains tissue fluid homeostasis, helps mediate afferent immune responses, and promotes cancer metastasis. To address the role microRNAs (miRNAs) play in the development and function of the lymphatic vascular system, we defined the in vitro miRNA expression profiles of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) and identified four BVEC signature and two LEC signature miRNAs. Their vascular lineage-specific expression patterns were confirmed in vivo by quantitative real-time PCR and in situ hybridization. Functional characterization of the BVEC signature miRNA miR-31 identified a novel BVEC-specific posttranscriptional regulatory mechanism that inhibits the expression of lymphatic lineage-specific transcripts in vitro. We demonstrate that suppression of lymphatic differentiation is partially mediated via direct repression of PROX1, a transcription factor that functions as a master regulator of lymphatic lineage-specific differentiation. Finally, in vivo studies of Xenopus and zebrafish demonstrated that gain of miR-31 function impaired venous sprouting and lymphatic vascular development, thus highlighting the importance of miR-31 as a negative regulator of lymphatic development. Collectively, our findings identify miR-31 is a potent regulator of vascular lineage-specific differentiation and development in vertebrates.

KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia.

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.

Lymphatic imaging in humans with near-infrared fluorescence.

While the lymphatic system is increasingly associated with diseases of prevalence, study of these diseases is difficult owing to the paucity of imaging techniques with the sensitivity and temporal resolution to discriminate lymphatic function. Herein, we review the known, pertinent features of the human lymphatic system in health and disease and set the context for a number of emerging studies that use near-infrared fluorescence imaging to non-invasively assess tumor draining lymphatic basins in cancer patients, intraoperatively guide resection of first draining lymph nodes, and to interrogate the difference between normal and aberrant lymphatic structure and function.

Prox1 physically and functionally interacts with COUP-TFII to specify lymphatic endothelial cell fate.

Specification of endothelial cell (EC) fate during vascular development is controlled by distinct key regulators. While Notch plays an essential role in induction of arterial phenotypes, COUP-TFII is required to maintain the venous EC identity. Homeodomain transcription factor Prox1 functions to reprogram venous ECs to lymphatic endothelial cells (LECs). Here, we report that the venous EC fate regulator COUP-TFII is expressed in LECs throughout development and physically interacts with Prox1 to form a stable complex in various cell types including LECs. We found that COUP-TFII functions as a coregulator of Prox1 to control several lineage-specific genes including VEGFR-3, FGFR-3, and neuropilin-1 and is required along with Prox1 to maintain LEC phenotype. Together, we propose that the physical and functional interactions of the 2 proteins constitute an essential part in the program specifying LEC fate and may provide the molecular basis for the hypothesis of venous EC identity being the prerequisite for LEC specification.