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

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

Chronic High-Fat Diet Impairs Collecting Lymphatic Vessel Function in Mice

Lymphatic vessels play an essential role in intestinal lipid uptake, and impairment of lymphatic vessel function leads to enhanced adipose tissue accumulation in patients with lymphedema and in genetic mouse models of lymphatic dysfunction. However, the effects of obesity on lymphatic function have been poorly studied. We investigated if and how adipose tissue accumulation influences lymphatic function. Using a lymphatic specific tracer, we performed in vivo near-infrared (NIR) imaging to assess the function of collecting lymphatic vessels in mice fed normal chow or high-fat diet (HFD). Histological and whole mount analyses were performed to investigate the morphological changes in initial and the collecting lymphatic vessels. HFD was associated with impaired collecting lymphatic vessel function, as evidenced by reduced frequency of contractions and diminished response to mechanostimulation. Moreover, we found a significant negative correlation between collecting lymphatic vessel function and body weight. Whole mount analyses showed an enlargement of contractile collecting lymphatic vessels of the hind limb. In K14-VEGF-C mice, HFD resulted in a reduced spreading of the tracer within dermal lymphatic vessels. These findings indicate that adipose tissue expansion due to HFD leads to a functional impairment of the lymphatic vasculature, predominantly in collecting lymphatic vessels.     

Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.     

Molecular mechanisms of lymphangiogenesis in development and cancer

The lymphatic system, also named the second vascular system, plays a critical role in tissue homeostasis and immunosurveillance. The past two decades of intensive research have led to the identification and detailed understanding of many molecular players and mechanisms regulating the formation of the lymphatic vasculature during embryonic development. Furthermore, clinical and experimental data clearly demonstrate that the formation of new lymphatic vessels by sprouting lymphangiogenesis from pre-existing lymphatic vessels, or by the de novo formation of lymphatic capillaries also occurs in various pathological conditions, such as cancer and organ transplant rejection, while lymphangiogenesis is non-functional in primary edema. In cancer, lymphatic vessels are one major gateway for invasive tumor cells to leave the primary tumor site and to establish distant organ metastasis. Therefore, the specific targeting of the lymphatic vasculature at the tumor site could be a promising approach to prevent metastasis formation.     

Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3

The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.     

A Transgenic Prox1-Cre-tdTomato Reporter Mouse for Lymphatic Vessel Research

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.     

Mutation of Threonine 34 in Mouse Podoplanin-Fc Reduces CLEC-2 Binding and Toxicity in Vivo While Retaining Anti-lymphangiogenic Activity

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.     

Myeloid Cells Contribute to Tumor Lymphangiogenesis

The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation.     

Vascular development in early human embryos and in teratomas derived from human embryonic stem cells

During early human embryonic development, blood vessels are stimulated to grow, branch, and invade developing tissues and organs. Pluripotent human embryonic stem cells (hESCs) are endowed with the capacity to differentiate into cells of blood and lymphatic vessels. The present study aimed to follow vasculogenesis during the early stages of developing human vasculature and to examine whether human neovasculogenesis within teratomas generated in SCID mice from hESCs follows a similar course and can be used as a model for the development of human vasculature. Markers and gene profiling of smooth muscle cells and endothelial cells of blood and lymphatic vessels were used to follow neovasculogenesis and lymphangiogenesis in early developing human embryos (4-8 weeks) and in teratomas generated from hESCs. The involvement of vascular smooth muscle cells in the early stages of developing human embryonic blood vessels is demonstrated, as well as the remodeling kinetics of the developing human embryonic blood and lymphatic vasculature. In teratomas, human vascular cells were demonstrated to be associated with developing blood vessels. Processes of intensive remodeling of blood vessels during the early stages of human development are indicated by the upregulation of angiogenic factors and specific structural proteins. At the same time, evidence for lymphatic sprouting and moderate activation of lymphangiogenesis is demonstrated during these developmental stages. In the teratomas induced by hESCs, human angiogenesis and lymphangiogenesis are relatively insignificant. The main source of blood vessels developing within the teratomas is provided by the murine host. We conclude that the teratoma model has only limited value as a model to study human neovasculogenesis and that other in vitro methods for spontaneous and guided differentiation of hESCs may prove more useful.     

A novel podoplanin‐GFPCre mouse strain for gene deletion in lymphatic endothelial cells

The lymphatic vascular system is a one‐direction network of thin‐walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5’ regulatory region. Pdpn encodes a transmembrane mucin‐type O‐glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs.     

Lymphangiogenesis in post-natal tissue remodeling: lymphatic endothelial cell connection with its environment

The main physiological function of the lymphatic vasculature is to maintain tissue fluid homeostasis. Lymphangiogenesis or de novo lymphatic formation is closely associated with tissue inflammation in adults (i.e. wound healing, allograft rejection, tumor metastasis). Until recently, research on lymphangiogenesis focused mainly on growth factor/growth factor-receptor pathways governing this process. One of the lymphatic vessel features is the incomplete or absence of basement membrane. This close association of endothelial cells with the underlying interstitial matrix suggests that cell–matrix interactions play an important role in lymphangiogenesis and lymphatic functions. However, the exploration of interaction between extracellular matrix (ECM) components and lymphatic endothelial cells is in its infancy. Herein, we describe ECM–cell and cell–cell interactions on lymphatic system function and their modification occurring in pathologies including cancer metastasis.     

Macrophages define dermal lymphatic vessel calibre during development by regulating lymphatic endothelial cell proliferation

Macrophages have been suggested to stimulate neo-lymphangiogenesis in settings of inflammation via two potential mechanisms: (1) acting as a source of lymphatic endothelial progenitor cells via the ability to transdifferentiate into lymphatic endothelial cells and be incorporated into growing lymphatic vessels; and (2) providing a crucial source of pro-lymphangiogenic growth factors and proteases. We set out to establish whether cells of the myeloid lineage are important for development of the lymphatic vasculature through either of these mechanisms. Here, we provide lineage tracing evidence to demonstrate that lymphatic endothelial cells arise independently of the myeloid lineage during both embryogenesis and tumour-stimulated lymphangiogenesis in the mouse, thus excluding macrophages as a source of lymphatic endothelial progenitor cells in these settings. In addition, we demonstrate that the dermal lymphatic vasculature of PU.1(-/-) and Csf1r(-/-) macrophage-deficient mouse embryos is hyperplastic owing to elevated lymphatic endothelial cell proliferation, suggesting that cells of the myeloid lineage provide signals that act to restrain lymphatic vessel calibre in the skin during development. In contrast to what has been demonstrated in settings of inflammation, macrophages do not comprise the principal source of pro-lymphangiogenic growth factors, including VEGFC and VEGFD, in the embryonic dermal microenvironment, illustrating that the sources of patterning and proliferative signals driving embryonic and disease-stimulated lymphangiogenesis are likely to be distinct.     

An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish

Background

Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.

Methods and Results

Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.

Conclusion

Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.     

Density of intranodal lymphatics and VEGF-C expression in B-cell lymphoma and reactive lymph nodes

Lymphatic vasculature in solid tumors may serve as the pathway for metastatic spread of the cancer to the regional lymph nodes and to distant organs. Controversy still exists whether tumors metastasize through existing lymphatics or through newly formed vessels (lymphangiogenesis). The role of lymphangiogenesis in lymphoma spread and proliferation is not clearly established. VEGF-C is the most potent inducer of lymphangiogenesis. LYVE-1 was shown to be a specific marker for lymphatic vessels in normal and tumor tissue. The aim of the present study was the evaluation of lymph node LYVE-1-positive lymphatic sinus density (LSD) and VEGF-C expression in patients with non-Hodgkin's lymphoma (nHL) and in reactive lymph nodes. Sixty paraffin-embedded lymph nodes from newly diagnosed patients with B-cell nHL were evaluated. Twelve lymph node biopsy specimens from adult patients with reactive lymphonodulitis were used as controls. Sections of lymph nodes were stained immunohistochemically for LYVE-1 and VEGF-C. VEGF-C expression in lymph nodes of nHL patients was low and not significantly different from that in the control (p = 0.6). Moreover, VEGF-C expression did not differ significantly between aggressive and indolent lymphomas (p = 0.53). Similarly we did not find differences in LSD in aggressive nHL and in indolent nHL (p=0.49). The mean LSD in reactive lymph nodes was higher than in nHL (p = 0.03). Only in 2 out of 12 reactive lymph nodes LYVE-1-positive vessels were absent. In all groups we demonstrated a strong positive correlation between VEGF-C and LYVE-1-expression (p = 0.0001). Higher LSD in reactive lymph nodes as compared to those of nHL patients suggests that lymphoma proliferation leads to the destruction of the existing lymphatics rather than to lymphangiogenesis within lymph nodes. NHL are not associated with increased expression of VEGF-C nor increased LYVE-1-positive lymphatic sinuses density within lymph nodes.     

Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos

Lymphatic vessels play an important role in the regulation of tissue fluid balance, immune responses, and fat adsorption and are involved in diseases including lymphedema and tumor metastasis. Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) is necessary for development of the blood vasculature during early embryogenesis, but later, VEGFR-3 expression becomes restricted to the lymphatic vasculature. We analyzed mice deficient in both of the known VEGFR-3 ligands, VEGF-C and VEGF-D. Unlike the Vegfr3^−/− embryos, the Vegfc^−/−; Vegfd^−/− embryos displayed normal blood vasculature after embryonic day 9.5. Deletion of Vegfr3 in the epiblast, using keratin 19 (K19) Cre, resulted in a phenotype identical to that of the Vegfr3^−/− embryos, suggesting that this phenotype is due to defects in the embryo proper and not in placental development. Interestingly, the Vegfr3^neo hypomorphic mutant mice carrying the neomycin cassette between exons 1 and 2 showed defective lymphatic development. Overexpression of human or mouse VEGF-D in the skin, under the K14 promoter, rescued the lymphatic hypoplasia of the Vegfc^+/− mice in the K14-VEGF-D; Vegfc^+/− compound mice, suggesting that VEGF-D is functionally redundant with VEGF-C in the stimulation of developmental lymphangiogenesis. Our results suggest VEGF-C- and VEGF-D-independent functions for VEGFR-3 in the early embryo.     

KDR-LacZ-expressing cells are involved in ovarian and testis-specific vascular development, suggesting a role for VEGFA in the regulation of this vasculature

Our objectives were to evaluate kinase insert domain protein receptor (KDR)-β-galactosidase (LacZ) expression as a marker for vascular development during gonadal morphogenesis and to determine whether any novel non-angiogenic KDR-LacZ expression was present in mouse testes or ovaries. Gonads were collected from mice expressing LacZ driven by the Kdr promoter (KDR-LacZ) from embryonic day 11 (E11) through postnatal day 60 (P60). At E11.5, mesonephric cells expressing KDR-LacZ seemed to migrate into the developing testis and surrounded developing seminiferous cords. Cells expressing KDR-LacZ appeared in the ovary with no apparent migration from the adjacent mesonephros, suggesting a different origin of endothelial cells. Testis organ cultures from E11 mice were treated with 8 μM VEGFR-TKI, a vascular endothelial growth factor A signal transduction inhibitor; subsequently, the amount of KDR-LacZ staining was reduced by 66%-99% (P<0.002), and the ability of KDR-expressing cells to form a densely organized vascular network was inhibited. Novel non-angiogenic KDR-LacZ staining was detected in the testis on specific subsets of germ cells at E16, E17, P4, P20, P30, and P60. In ovaries, staining was present on oocytes within oocyte cysts at E17 and within late secondary follicles of postnatal mice. Thus, KDR is an excellent marker for analyzing vascular development in the gonads. Inhibition of VEGFA signal transduction prevents the development of testis-specific vasculature. Furthermore, non-vascular KDR-LacZ staining suggests that KDR directly affects both spermatogenesis and somatic-oocyte interactions during gametogenesis.     

Protein Tyrosine Phosphatase PTPN14 Is a Regulator of Lymphatic Function and Choanal Development in Humans

The lymphatic vasculature is essential for the recirculation of extracellular fluid, fat absorption, and immune function and as a route of tumor metastasis. The dissection of molecular mechanisms underlying lymphangiogenesis has been accelerated by the identification of tissue-specific lymphatic endothelial markers and the study of congenital lymphedema syndromes. We report the results of genetic analyses of a kindred inheriting a unique autosomal-recessive lymphedema-choanal atresia syndrome. These studies establish linkage of the trait to chromosome 1q32-q41 and identify a loss-of-function mutation in PTPN14, which encodes a nonreceptor tyrosine phosphatase. The causal role of PTPN14 deficiency was confirmed by the generation of a murine Ptpn14 gene trap model that manifested lymphatic hyperplasia with lymphedema. Biochemical studies revealed a potential interaction between PTPN14 and the vascular endothelial growth factor receptor 3 (VEGFR3), a receptor tyrosine kinase essential for lymphangiogenesis. These results suggest a unique and conserved role for PTPN14 in the regulation of lymphatic development in mammals and a nonconserved role in choanal development in humans.     

EMILIN1/α9β1 Integrin Interaction Is Crucial in Lymphatic Valve Formation and Maintenance

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometric in vitro and in vivo functional assays, we conclude that this impaired phenotype depends on the lack of α9β1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a “guiding” molecule in migration of lymphatic endothelial cells.