Lymphatic Endothelial Cell Fate Research Articles

Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.

During development, the lymphatic vasculature forms as a second network derived chiefly from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is a key step in this process. Specification, differentiation and maintenance of LEC fate are all driven by thetranscription factor Prox1, yet the downstream mechanisms remain to be elucidated. We here present a single-cell transcriptomic atlas of lymphangiogenesis in zebrafish, revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single-cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a LEC-VEC fate shift. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new downstream targets and regulatory regions of the genome controlled by Prox1 and presents evidence that Prox1 specifies LEC fate primarily by limiting blood vascular and haematopoietic fate. This extensive single-cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance.

Recent findings indicate that mitochondrial respiration regulates blood endothelial cell proliferation; however, its role in differentiating lymphatic endothelial cells (LECs) is unknown. We hypothesized that mitochondria could work as a sensor of LECs' metabolic specific needs by determining their functional requirements according to their differentiation status and local tissue microenvironment. Accordingly, we conditionally deleted the QPC subunit of mitochondrial complex III in differentiating LECs of mouse embryos. Unexpectedly, mutant mice were devoid of a lymphatic vasculature by mid-gestation, a consequence of the specific down-regulation of main LEC fate regulators, particularly Vegfr3, leading to the loss of LEC fate. Mechanistically, this is a result of reduced H3K4me3 and H3K27ac in the genomic locus of key LEC fate controllers (e.g., Vegfr3 and Prox1). Our findings indicate that by sensing the LEC differentiation status and microenvironmental metabolic conditions, mitochondrial complex III regulates the critical Prox1-Vegfr3 feedback loop and, therefore, LEC fate specification and maintenance.

Beyond PROX1: transcriptional, epigenetic, and noncoding RNA regulation of lymphatic identity and function.

The lymphatic vascular system acts as the major transportation highway of tissue fluids, and its activation or impairment is associated with a wide range of diseases. There has been increasing interest in understanding the mechanisms that control lymphatic vessel formation (lymphangiogenesis) and function in development and disease. Here, we discuss recent insights into new players whose identification has contributed to deciphering the lymphatic regulatory code. We reveal how lymphatic endothelial cells, the building blocks of lymphatic vessels, utilize their transcriptional, post-transcriptional, and epigenetic portfolio to commit to and maintain their vascular lineage identity and function, with a particular focus on development.

Network patterning, morphogenesis and growth in lymphatic vascular development.

The lymphatic vasculature is a vital component of the vertebrate vascular system that mediates tissue fluid homeostasis, lipid uptake and immune surveillance. The development of the lymphatic vasculature starts in the early vertebrate embryo, when a subset of blood vascular endothelial cells of the cardinal veins acquires lymphatic endothelial cell fate. These cells sprout from the veins, migrate, proliferate and organize to give rise to a highly structured and unique vascular network. Cellular cross-talk, cell-cell communication and the interpretation of signals from surrounding tissues are all essential for coordinating these processes. In this chapter, we highlight new findings and review research progress with a particular focus on LEC migration and guidance, expansion of the LEC lineage, network remodeling and morphogenesis of the lymphatic vasculature.

Ras Pathways on Prox1 and Lymphangiogenesis: Insights for Therapeutics.

Over the past couple of decades, lymphatics research has accelerated and gained a much-needed recognition in pathophysiology. As the lymphatic system plays heavy roles in interstitial fluid drainage, immune surveillance and lipid absorption, the ablation or excessive growth of this vasculature could be associated with many complications, from lymphedema to metastasis. Despite their growing importance in cancer, few anti-lymphangiogenic therapies exist today, as they have yet to pass phase 3 clinical trials and acquire FDA approval. As such, many studies are being done to better define the signaling pathways that govern lymphangiogenesis, in hopes of developing new therapeutic approaches to inhibit or stimulate this process. This review will cover our current understanding of the Ras signaling pathways and their interactions with Prox1, the master transcriptional switch involved in specifying lymphatic endothelial cell fate and lymphangiogenesis, in hopes of providing insights to lymphangiogenesis-based therapies.

Oncogenic Herpesvirus Engages Endothelial Transcription Factors SOX18 and PROX1 to Increase Viral Genome Copies and Virus Production.

Kaposi sarcoma is a tumor caused by Kaposi sarcoma herpesvirus (KSHV) infection and is thought to originate from lymphatic endothelial cells (LEC). While KSHV establishes latency in virtually all susceptible cell types, LECs support spontaneous expression of oncogenic lytic genes, high viral genome copies, and release of infectious virus. It remains unknown the contribution of spontaneous virus production to the expansion of KSHV-infected tumor cells and the cellular factors that render the lymphatic environment unique to KSHV life cycle. We show here that expansion of the infected cell population, observed in LECs, but not in blood endothelial cells, is dependent on the spontaneous virus production from infected LECs. The drivers of lymphatic endothelium development, SOX18 and PROX1, regulated different steps of the KSHV life cycle. SOX18 enhanced the number of intracellular viral genome copies and bound to the viral origins of replication. Genetic depletion or chemical inhibition of SOX18 caused a decrease of KSHV genome copy numbers. PROX1 interacted with ORF50, the viral initiator of lytic replication, and bound to the KSHV genome in the promoter region of ORF50, increasing its transactivation activity and KSHV spontaneous lytic gene expression and infectious virus release. In Kaposi sarcoma tumors, SOX18 and PROX1 expression correlated with latent and lytic KSHV protein expression. These results demonstrate the importance of two key transcriptional drivers of LEC fate in the regulation of the tumorigenic KSHV life cycle. Moreover, they introduce molecular targeting of SOX18 as a potential novel therapeutic avenue in Kaposi sarcoma. SIGNIFICANCE: SOX18 and PROX1, central regulators of lymphatic development, are key factors for KSHV genome maintenance and lytic cycle in lymphatic endothelial cells, supporting Kaposi sarcoma tumorigenesis and representing attractive therapeutic targets.

Localised Collagen2a1 secretion supports lymphatic endothelial cell migration in the zebrafish embryo

The lymphatic vasculature develops primarily from pre-existing veins. A pool of lymphatic endothelial cells (LECs) first sprouts from cardinal veins followed by migration and proliferation to colonise embryonic tissues. Although much is known about the molecular regulation of LEC fate and sprouting during early lymphangiogenesis, we know far less about the instructive and permissive signals that support LEC migration through the embryo. Using a forward genetic screen, we identified mbtps1 and sec23a, components of the COP-II protein secretory pathway, as essential for developmental lymphangiogenesis. In both mutants, LECs initially depart the cardinal vein but then fail in their ongoing migration. A key cargo that failed to be secreted in both mutants was a type II collagen (Col2a1). Col2a1 is normally secreted by notochord sheath cells, alongside which LECs migrate. col2a1a mutants displayed defects in the migratory behaviour of LECs and failed lymphangiogenesis. These studies thus identify Col2a1 as a key cargo secreted by notochord sheath cells and required for the migration of LECs. These findings combine with our current understanding to suggest that successive cell-to-cell and cell-matrix interactions regulate the migration of LECs through the embryonic environment during development.

Lymphatic‐to‐blood vessel transition in adult microvascular networks: A discovery made possible by a top‐down approach to biomimetic model development

Emerging areas of vascular biology focus on lymphatic/blood vessel mispatterning and the regulation of endothelial cell identity. However, a fundamental question remains unanswered: Can lymphatic vessels become blood vessels in adult tissues? Leveraging a novel tissue culture model, the objective of this study was to track lymphatic endothelial cell fate over the time course of adult microvascular network remodeling. Cultured adult Wistar rat mesenteric tissues were labeled with BSI-lectin and time-lapse images were captured over five days of serum-stimulated remodeling. Additionally, rat mesenteric tissues on day 0 and day 3 and 5 post-culture were labeled for PECAM+LYVE-1 or PECAM+podoplanin. Cultured networks were characterized by increases in blood capillary sprouting, lymphatic sprouting, and the number of lymphatic/blood vessel connections. Comparison of images from the same network regions identified incorporation of lymphatic vessels into blood vessels. Mosaic lymphatic/blood vessels contained lymphatic marker positive and negative endothelial cells. Our results reveal the ability for lymphatic vessels to transition into blood vessels in adult microvascular networks and discover a new paradigm for investigating lymphatic/blood endothelial cell dynamics during microvascular remodeling.

Evolutionary Differences in the Vegf/Vegfr Code Reveal Organotypic Roles for the Endothelial Cell Receptor Kdr in Developmental Lymphangiogenesis

Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in anorgan-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to befully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis.

SOX18 mutations associated with human HLTRS syndrome reveal modification of protein‐protein interactions

Key transcription factors (TFs) including SRY‐related HMG box‐containing protein 18 (SOX18), COUP‐TFII, and prospero‐related homeobox 1 (PROX1) are responsible for the molecular control of the lymphatic endothelial cell fate. Mutations or truncations in the coding sequence of these TFs can cause a variety of human diseases. In particular, mutations in the transcription factor SOX18 cause the Hypotricosis‐Lymphedema‐Telangiectasia syndrome (HTLS), a congenital condition characterized for presenting swelling of tissue due to malformation or malfunction of lymphatics (Lymphedema), furthermore the people who present the disease also present alopecia (Hypotricosis) and the presence of small dilated vessels near the surface of the skin (Telangiectasia). Some patients also present with renal defect and hypertension, in a new variant of the disease called HLTRS (Hypotricosis‐Lymphedema‐Telangiectasia renal defect syndrome). So far two types of SOX18 variants have been described to be associated with HTLS: missense mutations that lack the transactivation domain and nonsense mutations, in the DNA binding domain. We aimed at identifying how the protein‐protein interactions (PPI) network is affected by these mutations and correlate interactions losses with phenotypic outcomes.We first established a SOX18 interactome, validating interaction with 31 SOX18 potential co‐factors. Then we characterized the stability of the mutated proteins by single‐molecule fluorescence and measured physical interactions using different in vitro techniques. Our data show that the SOX18 mutants are thermally stable and still able to bind DNA. However they are losing a significant number of protein partners, the extent and profile of these interaction losses was found to depend on the mutations. This preliminary data set is slowly providing a molecular view of the disease and is opening the door for potential pharmaceutical treatment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deregulation of HDAC5 by Viral Interferon Regulatory Factor 3 Plays an Essential Role in Kaposi's Sarcoma-Associated Herpesvirus-Induced Lymphangiogenesis.

ABSTRACTKaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi’s sarcoma (KS), which is one of the most common HIV-associated neoplasms. The endothelium is the thin layer of squamous cells where vascular blood endothelial cells (BECs) line the interior surface of blood vessels and lymphatic endothelial cells (LECs) are in direct contact with lymphatic vessels. The KS lesions contain a prominent compartment of neoplastic spindle morphology cells that are closely related to LECs. Furthermore, while KSHV can infect both LECs and BECs in vitro, its infection activates genetic programming related to lymphatic endothelial cell fate, suggesting that lymphangiogenic pathways are involved in KSHV infection and malignancy. Here, we report for the first time that viral interferon regulatory factor 3 (vIRF3) is readily detected in over 40% of KS lesions and that vIRF3 functions as a proangiogenic factor, inducing hypersprouting formation and abnormal growth in a LEC-specific manner. Mass spectrometry analysis revealed that vIRF3 interacted with histone deacetylase 5 (HDAC5), which is a signal-responsive regulator for vascular homeostasis. This interaction blocked the phosphorylation-dependent cytosolic translocation of HDAC5 and ultimately altered global gene expression in LECs but not in BECs. Consequently, vIRF3 robustly induced spindle morphology and hypersprouting formation of LECs but not BECs. Finally, KSHV infection led to the hypersprouting formation of LECs, whereas infection with a ΔvIRF3 mutant did not do so. Collectively, our data indicate that vIRF3 alters global gene expression and induces a hypersprouting formation in an HDAC5-binding-dependent and LEC-specific manner, ultimately contributing to KSHV-associated pathogenesis.

Lymphatic Endothelial Cell Plasticity in Development and Disease.

The lymphatic vasculature is crucial for maintaining tissue-fluid homeostasis, providing immune surveillance and mediating lipid absorption. The lymphatic vasculature is tightly associated with the blood vasculature, although it exhibits distinct morphological and functional features. Endothelial cells (ECs) lineage fate specification is determined during embryonic development; however, accumulating evidence suggests that differentiated ECs exhibit remarkable heterogeneity and plasticity. In this review, we provide an overview of the molecular mechanisms promoting lymphatic cell fate specification in the mammalian embryo. We also summarize available data suggesting that lymphatic EC fate is reprogrammable in normal and pathological settings. We further discuss the possible advantages of cell fate manipulation to treat certain disorders associated with lymphatic dysfunction.

Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function.

The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate.

Roles of transcriptional network during the formation of lymphatic vessels

The lymphatic vascular system, also known as the second vascular system in vertebrates, plays crucial roles in various physiological and pathological processes. It participates in the maintenance of normal tissue fluid balance, trafficking of the immune cells and absorption of fatty acids in the gut. Furthermore, lymphatic system is associated with the pathogenesis of a number of diseases, including lymphedema, inflammatory diseases and tumour metastasis. Lymphatic vessels are comprised of lymphatic endothelial cells (LECs), which are differentiated from blood vascular endothelial cells. This review highlights recent advances in our understanding of the transcriptional control of LEC fate determination and reflects on efforts to understand the roles of transcriptional networks during this discrete developmental process.

The Transcriptional Control of Lymphatic Vascular Development

More than 100 years ago, Florence Sabin suggested that lymphatic vessels develop by sprouting from preexisting blood vessels, but it is only over the past decade that the molecular mechanisms underpinning lymphatic vascular development have begun to be elucidated. Genetic manipulations in mice have identified a transcriptional hub comprised of Prox1, CoupTFII, and Sox18 that is essential for lymphatic endothelial cell fate specification. Recent work has identified a number of additional transcription factors that regulate later stages of lymphatic vessel differentiation and maturation. This review highlights recent advances in our understanding of the transcriptional control of lymphatic vascular development and reflects on efforts to better understand the activities of transcriptional networks during this discrete developmental process. Finally, we highlight the transcription factors associated with human lymphatic vascular disorders, demonstrating the importance of understanding how the activity of these key molecules is regulated, with a view toward the development of innovative therapeutic avenues.

Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein

The homeobox transcription factor PROX1 is essential for the development and maintenance of lymphatic vasculature. How PROX1 regulates lymphatic endothelial cell fate remains undefined. PROX1 has been shown to upregulate the expression of Cyclin E, which mediates the G 1 to S transition of the cell cycle. Here we demonstrate that PROX1 activates the mouse Cyclin E1 ( Ccne1) promoter via two proximal E2F-binding sites. We have determined that the N-terminal region of PROX1 is sufficient to activate a 1-kb Ccne1 promoter, whereas the homeodomain is dispensable for activation. We have identified that the Prospero domain 1 (PD1) is required for the nuclear localization of PROX1. Our comparison of two DNA-binding-deficient constructs of PROX1 showed a cell-type-specific difference between these two proteins in both their localization and function. We demonstrated that siRNA-mediated knockdown of PROX1 in lymphatic endothelial cells decreases progression from G 1 to S phase of the cell cycle. We conclude that PROX1 activates the Ccne1 promoter independent of DNA binding, and our results illustrate a novel role for PROX1 in the regulation of lymphatic endothelial cell proliferation.

Prox1 expression is negatively regulated by miR-181 in endothelial cells

AbstractThe specification of arterial, venous, and lymphatic endothelial cell fate is critical during vascular development. Although the homeobox transcription factor, Prox1, is crucial for the specification and maintenance of lymphatic endothelial cell identity, little is known regarding the mechanisms that regulate Prox1 expression. Here we demonstrate that miR-181a binds the 3′ untranslated region of Prox1, resulting in translational inhibition and transcript degradation. Increased miR-181a activity in primary embryonic lymphatic endothelial cells resulted in substantially reduced levels of Prox1 mRNA and protein and reprogramming of lymphatic endothelial cells toward a blood vascular phenotype. Conversely, treatment of primary embryonic blood vascular endothelial cells with miR-181a antagomir resulted in increased Prox1 mRNA levels. miR-181a expression is significantly higher in embryonic blood vascular endothelial cells compared with lymphatic endothelial cells, suggesting that miR-181 activity could be an important mechanism by which Prox1 expression is silenced in the blood vasculature during development. Our work is the first example of a microRNA that targets Prox1 and has implications for the control of Prox1 expression during vascular development and neo-lymphangiogenesis.

An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells

AbstractArteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may coreside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators.

Specification of arterial, venous, and lymphatic endothelial cells during embryonic development.

The groundbreaking discovery about arterial and venous expression of ephrinB2 and EphB4, respectively, in early embryonic development has led to a new paradigm for vascular research, providing compelling evidence that arterial and venous endothelial cells are established by genetic mechanisms before circulation begins. For arterial specification, vascular endothelial growth factor (VEGF) induces expression of Notch signaling genes, including Notch1 and its ligand, Delta-like 4 (Dll4), and Foxc1 and Foxc2 transcription factors directly regulate Dll4 expression. Upon activation of Notch signaling, the Notch downstream genes, Hey1/2 in mice or gridlock in zebrafish, further promote arterial differentiation. On the other hand, the orphan nuclear receptor COUP-TFII is a determinant factor for venous specification by inhibiting expression of arterial specific genes, including Nrp1 and Notch. After arterial and venous endothelial cells differentiate, a subpopulation of venous endothelial cells is thought to become competent to acquire lymphatic endothelial cell fate by progressively expressing the transcription factors Sox18 and Prox1 to differentiate into lymphatic endothelial cells. Therefore, it has now evident that arterial-venous cell fate determination and subsequent lymphatic development are regulated by the multi-step regulatory system associated with the key signaling pathways and transcription factors. Furthermore, new signaling molecules as additional regulators in these processes have recently been identified. As the mechanistic basis for a link between signaling pathways and transcriptional networks in arterial, venous and lymphatic endothelial cells begins to be uncovered, it is now time to summarize the literature on this exciting topic and provide perspectives for future research in the field.

Endothelial cell plasticity: how to become and remain a lymphatic endothelial cell

Lineage commitment and differentiation into mature cell types are mostly considered to be unidirectional and irreversible processes. However, recent results have challenged this by showing that terminally differentiated cell types can be reprogrammed into other cell types, an important step towards devising strategies for gene therapy and tissue regeneration. In this Review, we summarize recent data on the earliest steps in the development of the mammalian lymphatic vasculature: the specification of lymphatic endothelial cells (LECs). We elaborate on a developmental model that integrates the different steps leading to LEC differentiation and lymphatic network formation, discuss evidence that suggests that LEC fate is plastic, and consider the potentially far-reaching implications of the ability to convert one cell type into another.