Primary Human Lymphatic Endothelial Cells Research Articles

The Alzheimer's disease-linked protease BACE2 cleaves VEGFR3 and modulates its signaling.

The β-secretase β-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer's disease.

Calcitonin gene-related peptide and intermedin induce phosphorylation of p44/42 MAPK in primary human lymphatic endothelial cells in vitro

Calcitonin gene-related peptide (CGRP) and adrenomedullin 2/intermedin (AM2/IMD) play important roles in several pathologies, including cardiovascular disease, migraine and cancer. The efficacy of drugs targeting CGRP signalling axis for the treatment of migraine patients is sometimes offset by side effects (e.g. inflammation and microvascular complications, including aberrant neovascularisation in the skin). Recent studies using animal models implicate CGRP in lymphangiogenesis and lymphatic vessel function. However, whether CGRP or AM2/IMD can act directly on lymphatic endothelial cells is unknown. Here, we found that CGRP and AM2/IMD induced p44/42 MAPK phosphorylation in a time- and dose-dependent manner in primary human dermal lymphatic endothelial cells (HDLEC) in vitro, and thus directly affected these cells. These new findings reveal CGRP and AM2/IMD as novel regulators of LEC biology and warrant further investigation of their roles in the context of pathologies associated with lymphatic function in the skin and other organs, and therapies targeting CGRP signalling axis.

Quantitative proteomics reveals CLR interactome in primary human cells

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine and cancer. To date, the network of proteins interacting with CLR (“CLR interactome”) in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation (IP) utilising anti-human CLR antibody with label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) and quantitative in situ proximity ligation assay (PLA). By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4,902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ PLA). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.

Analysis of tumor infiltrating immune cells in Kaposi sarcoma lesions discovers shifts in macrophage populations.

Limited information exists about the types of immune cells present in Kaposi sarcoma (KS) lesions, especially in KS in the gastrointestinal tract. Using previously reported RNA-sequencing results from Kaposi sarcoma lesions in skin and gastrointestinal tract with normal matched tissues from the same patients at the same time, we investigated changes in lymphocytes in these tissues. We employed a computational method that determines changes in cell type distributions using KS lesion transcriptome data compared to a reference set of RNA expression patterns of purified immune cells. Since secreted cytokines and chemokines from KSHV-infected cells may influence the microenvironment of Kaposi sarcoma lesions, we performed cytokine profiling of conditioned media from KSHV-infected primary human dermal lymphatic endothelial cells. We also measured how this conditioned media altered the differentiation of macrophages in cell culture assays. These results suggested that factors in conditioned media from KSHV-infected endothelial cells promoted differentiation of a promonocytic cell line to proinflammatory macrophages.

Trametinib inhibits lymphatic vessel invasion of bone in a mouse model of Gorham-Stout disease.

Gorham-Stout disease (GSD) is a rare lymphatic anomaly that can be caused by somatic activating mutations in KRAS. This discovery has led investigators to suggest that MEK inhibitors could be a novel treatment for GSD. However, the effect of MEK inhibitors on bone disease in animal models of GSD has not been investigated. We recently reported that Osx-tTA;TetO-Vegfc mice exhibit a phenotype that resembles GSD. Osx-tTA;TetO-Vegfc mice overexpress VEGF-C in bone, which stimulates the development of lymphatic vessels in bone and the gradual loss of cortical bone. The objective of this study was to characterize the effect of trametinib, an FDA-approved MEK1/2 inhibitor, on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. Immunoblotting was performed to assess the effect of trametinib on VEGF-C-induced phosphorylation of ERK1/2, AKT, and S6 in primary human lymphatic endothelial cells (LECs). Prevention and intervention experiments were performed to determine the effect of trametinib on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. We found that trametinib blocked VEGF-C-induced phosphorylation of ERK1/2 in primary human LECs. We also found that trametinib prevented VEGF-C-induced lymphatic invasion of bone and cortical bone loss in Osx-tTA;TetO-Vegfc mice. Additionally, trametinib slowed the progression of disease in Osx-tTA;TetO-Vegfc mice with established disease. However, it did not reverse disease in Osx-tTA;TetO-Vegfc mice. Our results show trametinib impacts bone disease in Osx-tTA;TetO-Vegfc mice. These findings further support the testing of MEK inhibitors in patients with GSD and other RAS pathway-driven complex lymphatic anomalies with bone involvement.

Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

First person – Sarit Pal

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Sarit Pal is first author on ‘ Nuclear localization of histamine receptor 2 in primary human lymphatic endothelial cells’, published in BiO. Sarit conducted the research described in this article while a graduate research assistant in Dr Anatoliy Gashev's lab at Texas A&M University, Bryan, TX, USA. He is now a senior field application scientist at BICO LLC, Waltham, MA, USA, investigating histamine signalling in the regulation of lymphatic immunophysiology.

Nuclear localization of histamine receptor 2 in primary human lymphatic endothelial cells.

ABSTRACTHistamine exerts its physiological functions through its four receptor subtypes. In this work, we report the subcellular localization of histamine receptor 2 (H2R), a G protein-coupled receptor (GPCR), which is expressed in a wide variety of cell and tissue types. A growing number of GPCRs have been shown to be localized in the nucleus and contribute toward transcriptional regulation. In this study, for the first time, we demonstrate the nuclear localization of H2R in lymphatic endothelial cells. In the presence of its ligand, we show significant upregulation of H2R nuclear translocation kinetics. Using fluorescently tagged histamine, we explored H2R-histamine binding interaction, which exhibits a critical role in this translocation event. Altogether, our results highlight the previously unrecognized nuclear localization pattern of H2R. At the same time, H2R as a GPCR imparts many unresolved questions, such as the functional relevance of this localization, and whether H2R can contribute directly to transcriptional regulation and can affect lymphatic specific gene expression. H2R blockers are commonly used medications that recently have shown significant side effects. Therefore, it is imperative to understand the precise molecular mechanism of H2R biology. In this aspect, our present data shed new light on the unexplored H2R signaling mechanisms. This article has an associated First Person interview with the first author of the paper.

Engineering transplantable human lymphatic and blood capillary networks in a porous scaffold

Due to a relative paucity of studies on human lymphatic assembly in vitro and subsequent in vivo transplantation, capillary formation and survival of primary human lymphatic (hLEC) and blood endothelial cells (hBEC) ± primary human vascular smooth muscle cells (hvSMC) were evaluated and compared in vitro and in vivo. hLEC ± hvSMC or hBEC ± hvSMC were seeded in a 3D porous scaffold in vitro, and capillary percent vascular volume (PVV) and vascular density (VD)/mm2 assessed. Scaffolds were also transplanted into a sub-cutaneous rat wound with morphology/morphometry assessment. Initially hBEC formed a larger vessel network in vitro than hLEC, with interconnected capillaries evident at 2 days. Interconnected lymphatic capillaries were slower (3 days) to assemble. hLEC capillaries demonstrated a significant overall increase in PVV (p = 0.0083) and VD (p = 0.0039) in vitro when co-cultured with hvSMC. A similar increase did not occur for hBEC + hvSMC in vitro, but hBEC + hvSMC in vivo significantly increased PVV (p = 0.0035) and VD (p = 0.0087). Morphology/morphometry established that hLEC vessels maintained distinct cell markers, and demonstrated significantly increased individual vessel and network size, and longer survival than hBEC capillaries in vivo, and established inosculation with rat lymphatics, with evidence of lymphatic function. The porous polyurethane scaffold provided advantages to capillary network formation due to its large (300–600 μm diameter) interconnected pores, and sufficient stability to ensure successful surgical transplantation in vivo. Given their successful survival and function in vivo within the porous scaffold, in vitro assembled hLEC networks using this method are potentially applicable to clinical scenarios requiring replacement of dysfunctional or absent lymphatic networks.

Loss of Primary Cilia Protein IFT20 Dysregulates Lymphatic Vessel Patterning in Development and Inflammation.

Microenvironmental signals produced during development or inflammation stimulate lymphatic endothelial cells to undergo lymphangiogenesis, in which they sprout, proliferate, and migrate to expand the vascular network. Many cell types detect changes in extracellular conditions via primary cilia, microtubule-based cellular protrusions that house specialized membrane receptors and signaling complexes. Primary cilia are critical for receipt of extracellular cues from both ligand-receptor pathways and physical forces such as fluid shear stress. Here, we report the presence of primary cilia on immortalized mouse and primary adult human dermal lymphatic endothelial cells in vitro and on both luminal and abluminal domains of mouse corneal, skin, and mesenteric lymphatic vessels in vivo. The purpose of this study was to determine the effects of disrupting primary cilia on lymphatic vessel patterning during development and inflammation. Intraflagellar transport protein 20 (IFT20) is part of the transport machinery required for ciliary assembly and function. To disrupt primary ciliary signaling, we generated global and lymphatic endothelium-specific IFT20 knockout mouse models and used immunofluorescence microscopy to quantify changes in lymphatic vessel patterning at E16.5 and in adult suture-mediated corneal lymphangiogenesis. Loss of IFT20 during development resulted in edema, increased and more variable lymphatic vessel caliber and branching, as well as red blood cell-filled lymphatics. We used a corneal suture model to determine ciliation status of lymphatic vessels during acute, recurrent, and tumor-associated inflammatory reactions and wound healing. Primary cilia were present on corneal lymphatics during all of the mechanistically distinct lymphatic patterning events of the model and assembled on lymphatic endothelial cells residing at the limbus, stalk, and vessel tip. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute corneal lymphangiogenesis resulting in increased lymphatic vessel density and branching. These data are the first functional studies of primary cilia on lymphatic endothelial cells and reveal a new dimension in regulation of lymphatic vascular biology.

Steroid hormone bioavailability is controlled by the lymphatic system

The steroid hormone progesterone accounts for immune tolerance in pregnancy. Enhanced progesterone metabolism to 6α-OH-pregnanolone occurs in complicated pregnancies such as in preeclampsia with preterm delivery or intrauterine growth restriction, and in cancer. As lymphatic endothelial cells (LECs) promote tumor immunity, we hypothesized that human LECs modify progesterone bioavailability. Primary human LECs and mice lymph nodes were incubated with progesterone and progesterone metabolism was analyzed by thin layer chromatography and liquid chromatography-mass spectrometry. Expression of steroidogenic enzymes, down-stream signal and steroid hormone receptors was assessed by Real-time PCR. The placental cell line HTR-8/SV neo was used as reference. The impact of the progesterone metabolites of interest was investigated on the immune system by fluorescence-activated cell sorting analysis. LECs metabolize progesterone to 6α-OH-pregnanolone and reactivate progesterone from a precursor. LECs highly express 17β-hydroxysteroid dehydrogenase 2 and are therefore antiandrogenic and antiestrogenic. LECs express several steroid hormone receptors and PIBF1. Progesterone and its metabolites reduced TNF-α and IFN-γ production in CD4+ and CD8+ T cells. LECs modify progesterone bioavailability and are a target of steroid hormones. Given the global area represented by LECs, they might have a critical immunomodulatory control in pregnancy and cancer.

An Approach to Study Melanoma Invasion and Crosstalk with Lymphatic Endothelial Cell Spheroids in 3D Using Immunofluorescence.

Three-dimensional (3D) cell culture has allowed a deeper understanding of complex pathological and physiological processes, overcoming some of the limitations of 2D cell culture on plastic and avoiding the costs and ethical issues related to experiments involving animals. Here we describe a protocol to embed single melanoma cells alone or together with primary human lymphatic endothelial cells in a 3D cross-linked matrix, to investigate the invasion and molecular crosstalk between these two cell types, respectively. After fixation and staining with antibodies and fluorescent conjugates, phenotypic changes in both cell types can be specifically analyzed by confocal microscopy.

Epigenetic regulation of the lineage specificity of primary human dermal lymphatic and blood vascular endothelial cells

Lymphatic and blood vascular endothelial cells (ECs) share several molecular and developmental features. However, these two cell types possess distinct phenotypic signatures, reflecting their different biological functions. Despite significant advances in elucidating how the specification of lymphatic and blood vascular ECs is regulated at the transcriptional level during development, the key molecular mechanisms governing their lineage identity under physiological or pathological conditions remain poorly understood. To explore the epigenomic signatures in the maintenance of EC lineage specificity, we compared the transcriptomic landscapes, histone composition (H3K4me3 and H3K27me3) and DNA methylomes of cultured matched human primary dermal lymphatic and blood vascular ECs. Our findings reveal that blood vascular lineage genes manifest a more ‘repressed’ histone composition in lymphatic ECs, whereas DNA methylation at promoters is less linked to the differential transcriptomes of lymphatic versus blood vascular ECs. Meta-analyses identified two transcriptional regulators, BCL6 and MEF2C, which potentially govern endothelial lineage specificity. Notably, the blood vascular endothelial lineage markers CD34, ESAM and FLT1 and the lymphatic endothelial lineage markers PROX1, PDPN and FLT4 exhibited highly differential epigenetic profiles and responded in distinct manners to epigenetic drug treatments. The perturbation of histone and DNA methylation selectively promoted the expression of blood vascular endothelial markers in lymphatic endothelial cells, but not vice versa. Overall, our study reveals that the fine regulation of lymphatic and blood vascular endothelial transcriptomes is maintained via several epigenetic mechanisms, which are crucial to the maintenance of endothelial cell identity.

Lymphatic Vessel on a Chip with Capability for Exposure to Cyclic Fluidic Flow.

The lymphatic system is a complex organ system that is essential in regulating the development of host immune responses. Because of the complexity of the lymphatic system and the existence of few in vitro models that replicate human lymphatic vessels, there is a need for a primary cell-based lymphatic model that can provide a better understanding of the effects of flow parameters, therapeutics, and other stimuli on lymphatic vessel behavior. In this report, a fluidic device models the cyclical lymphatic flow under normal and disease conditions. The device utilizes a pumpless design, operating with gravitational forces to simulate normal conditions with a shear of 0.092 Pa (0.92 dyn/cm2) as well as disease conditions with an increased shear of (0.67 Pa, 6.7 dyn/cm2). The cyclical pumping present in lymphatic vessels is replicated by applying shear stress for a period of 10 s multiple times per minute. Primary human lymphatic endothelial cells (HLECs) cultured in the device for 10 days produce less interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α) per cell than cells cultured under static conditions. The results are consistent with previously published in vivo measurements, indicating that the fluidic device mimics conditions for IL-8 and TNF-α expression well. Data obtained with the devices also indicate that primary HLECs proliferate faster under high-shear than under low-shear conditions.

KSHV requires vCyclin to overcome replicative senescence in primary human lymphatic endothelial cells.

Kaposi’s Sarcoma Herpesvirus (KSHV) is present in the main tumor cells of Kaposi’s Sarcoma (KS), the spindle cells, which are of endothelial origin. KSHV is also associated with two B-cell lymphomas, Primary Effusion Lymphoma (PEL) and Multicentric Castleman’s Disease. In KS and PEL, KSHV is primarily latent in the infected cells, expressing only a few genes. Although KSHV infection is required for KS and PEL, it is unclear how latent gene expression contributes to their formation. Proliferation of cancer cells occurs despite multiple checkpoints intended to prevent dysregulated cell growth. The first of these checkpoints, caused by shortening of telomeres, results in replicative senescence, where cells are metabolically active, but no longer divide. We found that human dermal lymphatic endothelial cells (LECs) are more susceptible to KSHV infection than their blood-specific endothelial cell counterparts and maintain KSHV latency to higher levels during passage. Importantly, KSHV infection of human LECs but not human BECs promotes their continued proliferation beyond this first checkpoint of replicative senescence. The latently expressed viral cyclin homolog is essential for KSHV-induced bypass of senescence in LECs. These data suggest that LECs may be an important reservoir for KSHV infection and may play a role during KS tumor development and that the viral cyclin is a critical oncogene for this process.

Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity.

The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis remains obscure. Here, we show that several M. tuberculosis clinical isolates form intracellular cords in primary human lymphatic endothelial cells (hLECs) in vitro and in the lymph nodes of patients with tuberculosis. We identified via RNA-Seq a transcriptional program that activated, in infected-hLECs, cell survival and cytosolic surveillance of pathogens pathways. Consistent with this, cytosolic access was required for intracellular M. tuberculosis cording. Mycobacteria lacking ESX-1 type VII secretion system or phthiocerol dimycocerosates expression, which failed to access the cytosol, were indeed unable to form cords within hLECs. Finally, we show that M. tuberculosis cording is a size-dependent mechanism used by the pathogen to avoid its recognition by cytosolic sensors and evade either resting or IFN-γ-induced hLEC immunity. These results explain the long-standing association between M. tuberculosis cording and virulence and how virulent mycobacteria use intracellular cording as strategy to successfully adapt and persist in the lymphatic tracts.

Characterization of de novo lytic infection of dermal lymphatic microvascular endothelial cells by Kaposi's sarcoma-associated herpesvirus

The biology of primary lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection is still not well understood, which is largely attributed to the lack of cell lines permissive to robust lytic KSHV infection in vitro. Our study demonstrates that primary human dermal lymphatic microvascular endothelial cells (HDLMEC) support lytic KSHV replication following de novo infection, resulting in robust KSHV production, indicating that HDLMECs are suitable for studying the regulation of primary lytic KSHV infection. Importantly, by utilizing lytically infected HDLMECs, we show for the first time that the KSHV latent genes LANA and viral cyclin are required for lytic replication during de novo lytic infection, a function of these latent genes that has not yet been recognized. Since Kaposi's sarcoma is considered to be originated from infected lymphatic endothelial cells, HDLMECs represent a valuable in vitro cell culture model for investigating lytic KSHV infection, which has been understudied in KSHV pathogenesis.

TNF-derived peptides inhibit tumour growth and metastasis through cytolytic effects on tumour lymphatics.

Tumour necrosis factor (TNF) is a multi-functional cytokine with profound and diverse effects on physiology and pathology. Identifying the molecular determinants underlying the functions and pathogenic effects of TNF is key to understanding its mechanisms of action and identifying new therapeutic opportunities based on this important molecule. Previously, we showed that some evolutionarily conserved peptides derived from TNF could induce cell death (e.g. apoptosis and/or necrosis), a feature of immune defence mechanisms shared by many vertebrates. In this study, we demonstrated that necrosis-inducing peptide P16 kills human glioblastoma cancer cells and primary human hepatoma or renal cancer cells isolated from patients who had not responded to standard treatments. Importantly, we show that the necrosis-inducing peptide P1516 significantly improves survival by inhibiting tumour metastasis in a 4T1 breast cancer syngeneic graft mouse model. Because the lymphatic system is an important metastatic route in many cancers, we also tested the effect of TNF-derived peptides on monolayers of primary human lymphatic endothelial cells (hDLEC) and found that they increased junctional permeability by inducing cytoskeletal reorganization, gap junction formation and cell death. Transmission electron microscopy imaging evidence, structural analysis and in-vitro liposome leakage experiments strongly suggest that this killing is due to the cytolytic nature of these peptides. P1516 provides another example of a pro-cytotoxic TNF peptide that probably functions as a cryptic necrotic factor released by TNF degradation. Its ability to inhibit tumour metastasis and improve survival may form the basis of a novel approach to cancer therapy.

Cell Type-Specific Interferon-\u03b3-mediated Antagonism of KSHV Lytic Replication

Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally associated with several malignant tumors: Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL). KS remains the most common AIDS-related malignancy since the AIDS epidemic and thus has been extensively studied. KS is characterized as an angioproliferative disease with massive immune cell infiltration at the early stage. High levels of proinflammatory cytokines and growth factors are found in KS lesions, and their involvement in the survival and growth of tumor cells has been well characterized. However, little is known about the role of the inflammatory microenvironment in the regulation of KSHV gene expression and/or viral replication. In the present study, we demonstrated that IFN-γ and TNF-α profoundly inhibited KSHV progeny production in primary human lymphatic endothelial cells (LECs) as well as induced KSHV-producer cells (iSLK.219) with doxycycline. Of note, IFN-γ inhibited overall KSHV gene expression, while the effects of TNF-α were confined to a selected set of genes, which were also downregulated by IFN-γ. The addition of IFN-γ up to 36 hr after induction of viral lytic replication was effective in terms of the inhibition of infectious virion production, suggesting that its inhibitory effect is exerted at the early stages of KSHV life cycle. We believe these data have potentially important implications for rationalizing a therapeutic agent to treat KSHV-induced tumors in which lytic replication plays a critical role in their pathogenesis: KS and MCD.

GATA2 controls lymphatic endothelial cell junctional integrity and lymphovenous valve morphogenesis through miR-126.

ABSTRACTMutations in the transcription factor GATA2 cause lymphedema. GATA2 is necessary for the development of lymphatic valves and lymphovenous valves, and for the patterning of lymphatic vessels. Here, we report that GATA2 is not necessary for valvular endothelial cell (VEC) differentiation. Instead, GATA2 is required for VEC maintenance and morphogenesis. GATA2 is also necessary for the expression of the cell junction molecules VE-cadherin and claudin 5 in lymphatic vessels. We identified miR-126 as a target of GATA2, and miR-126−/− embryos recapitulate the phenotypes of mice lacking GATA2. Primary human lymphatic endothelial cells (HLECs) lacking GATA2 (HLECΔGATA2) have altered expression of claudin 5 and VE-cadherin, and blocking miR-126 activity in HLECs phenocopies these changes in expression. Importantly, overexpression of miR-126 in HLECΔGATA2 significantly rescues the cell junction defects. Thus, our work defines a new mechanism of GATA2 activity and uncovers miR-126 as a novel regulator of mammalian lymphatic vascular development.