Lymphatic Endothelial Cells Populations Research Articles

New reporter zebrafish line unveils heterogeneity among lymphatic endothelial cells during development.

In zebrafish, lymphatic endothelial cells (LECs) originate from multiple/several distinct progenitor populations and generate organ-specific lymphatic vasculatures. Cell fate and tissue specificities were determined using a combination of genetically engineered transgenic lines in which the promoter of a LEC-specific gene drives expression of a fluorescent reporter protein. We established a novel zebrafish transgenic line expressing eGFP under the control of part of the zebrafish batf3 promoter (Basic Leucine Zipper ATF-Like Transcription Factor 3). Spatiotemporal examination of Tg(batf3MIN:eGFP) transgenic fish revealed a typical lymphatic expression pattern, which does not perfectly recapitulate the expression pattern of existing LEC transgenic lines. eGFP+ cells constitute a heterogeneous endothelial cell population, which expressed LEC and/or blood endothelial cells (BEC) markers in different tissues. In addition, we characterize the renal eGFP+ cell as a population of interest to study kidney diseases and regeneration. Our Tg(batf3MIN:eGFP) reporter zebrafish line provides a useful system to study LEC populations, of which heterogeneity depends on origin of progenitors, tissue environment and physiological conditions. We further developed a novel fish-adapted tissue clearing method, which allows deep imaging and 3D-visualization of vascular and lymphatic networks in the whole organism.

Linkage of Metabolic Defects to Activated PIK3CA Alleles in Endothelial Cells Derived from Lymphatic Malformation.

Lymphatic endothelial cells (LECs) derived from lymphatic malformations (LMs) bear activated PIK3CA alleles yet display an inflammatory gene expression profile. A basis for the inflammatory phenotype was sought by screening for coexisting somatic mutations. Fourteen independent LEC populations bearing activated PIK3CA alleles were isolated from LM. These were characterized by the expression of growth and inflammatory genes (VEGFC, IL-6, COX-2, IL-8, HO-1, E-SEL) by qRT-PCR. Most commonly upregulated gene products were VEGFC, COX2, HO-1, and ANGPTL4. The specific inhibition of PI3K reduced VEGFC expression without resolving inflammation. Whole exome sequencing of six LM-LEC populations identified five novel somatically acquired alleles coexisting with activated PIK3CA alleles. Two affected genes regulate lipid droplet metabolism (FITM2 and ATG2A), two are gene regulators (MTA1 and TAF1L), and the fifth is an isoform of ANK3 (an endosomal/lysosomal protein). Inhibition of AMPK implicated its involvement in regulating COX-2 and HO-1 overexpression. ANGPTL4 expression was independent of AMPK and PI3K activity and reflected lipid stress demonstrated in normal LECs. AMPK activation with AICAR had a selective growth-limiting effect in a subset of LM-LEC isolates. Inflammatory stress displayed by LM-LECs is consistent with errors in lipid metabolism that may be linked to acquired mutations. The acquisition of PIK3CA alleles may be a permissive event that antagonizes inflammation and metabolic defect.

Migration of lymphatic endothelial cells and lymphatic vascular development in the craniofacial region of embryonic mice.

Lymphatic development in mice is initiated in the trunk at embryonic day (E) 9.5. This study aimed to examine the origin of craniofacial lymphatic endothelial cells (LECs) and the developmental process of lymphatic vessels in the mouse craniofacial region. Serial sections from ICR mouse embryos at E9.5-E14.5 were immunolabeled with LEC and venous endothelial cell (VEC) markers. These markers included prospero homeobox protein 1 (Prox1), vascular endothelial growth factor receptor 3 (Vegfr3), lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and C-C motif chemokine 2 (Ccl21) for LEC, and COUP transcription factor 2 (CoupTF2) and endomucin (Emcn) for VEC. LECs were monitored as an index in Prox1/Vegfr3 double-positive cells using three-dimensional analysis because LECs express Prox1 and Vegfr3 ab initio during lymphatic vascular development. LECs appeared in VECs of the lateral walls of cardinal veins (CVs) at E9.5. These LECs were dichotomized into LEC populations that formed lymph sacs close to CVs and were scattered in the surrounding CVs. The scattered LECs formed cellular streams and extended from the trunk to the mandibular arches at E10.5 - E11.5. In the mandibular arches, individual LECs aggregated, and formed lymph sacs and tubular lymphatic vessels at E11.5-E14.5. Expression of the LEC marker proteins Lyve1 and Ccl21 in LECs changed during craniofacial lymphatic vascular development. Collectively, these findings suggest that craniofacial LECs originate from CVs of the trunk and migrate into the mandibular arches. Additionally, we found that craniofacial lymphatic vessels are formed according to morphogenesis of individual LECs that migrate from CVs.

Engineering Blood and Lymphatic Microvascular Networks in Fibrin Matrices.

Vascular network engineering is essential for nutrient delivery to tissue-engineered constructs and, consequently, their survival. In addition, the functionality of tissues also depends on tissue drainage and immune cell accessibility, which are the main functions of the lymphatic system. Engineering both the blood and lymphatic microvasculature would advance the survival and functionality of tissue-engineered constructs. The aim of this study was to isolate pure populations of lymphatic endothelial cells (LEC) and blood vascular endothelial cells (BEC) from human dermal microvascular endothelial cells and to study their network formation in our previously described coculture model with adipose-derived stromal cells (ASC) in fibrin scaffolds. We could follow the network development over a period of 4 weeks by fluorescently labeling the cells. We show that LEC and BEC form separate networks, which are morphologically distinguishable and sustainable over several weeks. In addition, lymphatic network development was dependent on vascular endothelial growth factor (VEGF)-C, resulting in denser networks with increasing VEGF-C concentration. Finally, we confirm the necessity of cell–cell contact between endothelial cells and ASC for the formation of both blood and lymphatic microvascular networks. This model represents a valuable platform for in vitro drug testing and for the future in vivo studies on lymphatic and blood microvascularization.

Lymphatic endothelial lineage assemblage during corneal lymphangiogenesis

Postnatal inflammatory lymphangiogenesis presumably requires precise regulatory processes to properly assemble proliferating lymphatic endothelial cells (LECs). The specific mechanisms that regulate the assembly of LECs during new lymphatic vessel synthesis are unclear. Dynamic endothelial shuffling and rearrangement has been proposed as a mechanism of blood vessel growth. We developed genetic lineage-tracing strategies using an inductive transgenic technology to track the fate of entire tandem dimer tomato-positive (tdT) lymphatic vessels or small, in some cases clonal, populations of LECs. We coupled this platform with a suture-induced mouse model of corneal lymphangiogenesis and used different analytic microscopy techniques including serial live imaging to study the spatial properties of proliferating tdT+ LEC progenies. LEC precursors and their progeny expanded from the corneal limbal lymphatic vessel and were assembled contiguously to comprise a subunit within a new lymphatic vessel. VE-cadherin blockade induced morphologic abnormalities in newly synthesized lymphatic vessels, but did not disrupt the tdT+ lymphatic endothelial lineage assembly. Analysis of this static and dynamic data based largely on direct in vivo observations supports a model of lymphatic endothelial lineage assemblage during corneal inflammatory lymphangiogenesis.

A microarray analysis of two distinct lymphatic endothelial cell populations.

We have recently identified lymphatic endothelial cells (LECs) to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT) of LECs resulted in enrichment of the podoplaninhigh cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510) and describe how we analyzed the data to identify differently expressed genes in these two LEC populations.

Isolation, characterization, and functional analysis of ferret lymphatic endothelial cells

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases – the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models.

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

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

Mapping the distinctive populations of lymphatic endothelial cells in different zones of human lymph nodes.

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

Role of Lymphangiogenesis in Cancer

Regional lymph node metastasis is a common event in solid tumors and is considered a marker for dissemination, increased stage, and worse prognosis. Despite rapid advances in tumor biology, the molecular processes that underpin lymphatic invasion and lymph node metastasis remain poorly understood. However, exciting discoveries have been made in the field of lymphangiogenesis in recent years. The identification of vascular endothelial growth factor ligands and cognate receptors involved in lymphangiogenesis, an understanding of the embryology of the mammalian lymphatic system, the recent isolation of pure populations of lymphatic endothelial cells, the investigation of lymphatic metastases in animal models, and the identification of markers that discriminate lymphatics from blood vessels at immunohistochemistry are current advances in the field of lymphangiogenesis, and as such are the main focus of this article. This review also evaluates evidence for lymphangiogenesis (ie, new lymphatic vessel formation in cancer) and critically reviews current data on the prognostic significance of lymphatic vascular density in tumors. A targeted approach to block pathways of lymphangiogenesis seems to be an attractive anticancer treatment strategy. Conversely, promotion of lymphangiogenesis may be a promising approach to the management of treatment-induced lymphedema in cancer survivors. Finally, the implications of these developments in cancer therapeutics and directions for future research are discussed.

Lymphatic endothelium: An important interactive surface for malignant cells

Endothelial cells line the vessels which transport fluid and cells throughout the body. Although much attention has been paid to these cells in the context of the blood vascular system, endothelial cells also line lymphatic vessels. Recent progress in identifying growth factors which drive the development of lymphatic vessels and molecular markers specific for lymphatics has expanded our understanding of the role the lymphatic system plays in human pathology. Techniques for purifying populations of lymphatic endothelial cells also allow the in vitro analysis of this unique surface to explore its role in tumour metastasis, immune cell function and fluid transport. This review provides a synopsis of the recent data pertaining to the purification and culture of lymphatic endothelial cells, and the interaction of tumour cells with lymphatic endothelium.