Embryonic Lymphatic Development Research Articles

The YAP–TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer

The secreted protein Collagen and calcium-binding EGF Domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFβ-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in CRC patients. YAP/TAZ/TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts (CAFs), which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells (HLECs) in vitro and lymphangiogenesis in a CRC cell-derived xenograft (CDX) model in vivo. In addition, the bromodomain and extra-terminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ/TEAD4/BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.

Comprehensive elucidation of developmental functions of arachidonic acid-derived lipid mediators in zebrafish.

Prostaglandins (PGs) and leukotrienes (LTs) are major physiologically active lipid, which are biosynthesized from arachidonic acid (AA). AA is released from membrane phospholipids by phospholipase A2 (PLA2), and thus PGs and LTs are derived from membrane phospholipids. Zebrafish is a vertebrate model organism that has been widely used in the studies on development and drug discovery for the reasons as follows: (i) embryo development occurs outside the maternal body, (ii) embryo is transparent, (iii) high conservation between human and zebrafish genes, etc. Nevertheless, the systematic identification and characterization of zebrafish PG and LT receptors have not been performed. Therefore, we identified twelve zebrafish PG receptors and characterized their pharmacological properties. Furthermore, to identify the physiological role of zebrafish PG receptors, we generated each receptor-deficient zebrafish, and analyzed developmental processes. Here, we report that PGE2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Using pharmacological and genetic approaches, we demonstrated that the COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of critical factors for the lymphatic specification. We recently focused the zebrafish cysteinyl LT (cysLT) receptors, cloned four zebrafish cysLT receptors, and analyzed the expression profile of these receptors in developmental stages. As results, we revealed that the four zebrafish cysLT receptors show unique tissue distribution patterns. Each receptor may hence be involved in unique physiological functions. This work provides further insights into the diverse functions of arachidonate metabolites in zebrafish.

IL-1β Impacts Vascular Integrity and Lymphatic Function in the Embryonic Omentum.

The chromatin-remodeling enzyme BRG1 (brahma-related gene 1) regulates gene expression in a variety of rapidly differentiating cells during embryonic development. However, the critical genes that BRG1 regulates during lymphatic vascular development are unknown. We used genetic and imaging techniques to define the role of BRG1 in murine embryonic lymphatic development, although this approach inadvertently expanded our study to multiple interacting cell types. We found that omental macrophages fine-tune an unexpected developmental process by which erythrocytes escaping from naturally discontinuous omental blood vessels are collected by nearby lymphatic vessels. Our data indicate that circulating fibrin(ogen) leaking from gaps in omental blood vessels can trigger inflammasome-mediated IL-1β (interleukin-1β) production and secretion from nearby macrophages. IL-1β destabilizes adherens junctions in omental blood and lymphatic vessels, contributing to both extravasation of erythrocytes and their uptake by lymphatics. BRG1 regulates IL-1β production in omental macrophages by transcriptionally suppressing the inflammasome trigger RIPK3 (receptor interacting protein kinase 3). Genetic deletion of Brg1 in embryonic macrophages leads to excessive IL-1β production, erythrocyte leakage from blood vessels, and blood-filled lymphatics in the developing omentum. Altogether, these results highlight a novel context for epigenetically regulated crosstalk between macrophages, blood vessels, and lymphatics.

The novel miR-1269b-regulated protein SVEP1 induces hepatocellular carcinoma proliferation and metastasis likely through the PI3K/Akt pathway

Decreased intercellular adhesion is a key step in the metastasis and recurrence of many cancers, including hepatocellular carcinoma (HCC). SVEP1 is an important cell adhesion molecule that plays a key role in regulating intercellular adhesion and embryonic lymphatic development. However, the expression patterns and roles of SVEP1 in HCC are still largely unknown. We identified SVEP1 expression by analyzing 220 HCC samples from our cancer center. TCGA and GEO online-databases were used for data calibration and validation. SVEP1 was differentially expressed in two groups of HCCs with different risks of recurrence and was deemed as an independent risk factor for the prognosis of HCC. The expression of SVEP1 is negatively related to the proliferation and metastasis of HCC. Downregulation of SVEP1 expression promoted in vitro HCC cell migration, chemotaxis, invasion and proliferation, as well as in vivo tumor growth, local invasion and metastasis in a mouse model. Bioinformatic analysis and RT-PCR results showed that miR-1269b expression is negatively correlated with the SVEP1 expression and the prognosis of HCC patients. Further experiments showed that miR-1269b directly targets and downregulates the expression of SVEP1, which further induces the phosphorylation of Akt at thr308. These regulatory effects ultimately mediate the proliferation and metastasis of HCC cells. SVEP1 could serve as a promising prognostic marker of HCC. MiR-1269b downregulates SVEP1 expression and promotes HCC proliferation and metastasis likely through the PI3k/Akt signaling pathway.

Essential role of prostaglandin E2 and the EP3 receptor in lymphatic vessel development during zebrafish embryogenesis

Lymphatic endothelial cells arise from the venous endothelial cells in embryonic lymphatic development. However, the molecular mechanisms remain to be elucidated. We here report that prostaglandin (PG) E2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Knockdown of the EP3 receptor or inhibition of cyclooxygenases (COX; rate-limiting enzymes for PG synthesis) impaired lymphatic development by perturbing lymphatic specification during zebrafish development. These impairments by COX inhibition were recovered by treatment with sulprostone (EP1/3 agonist). Knockdown of the EP3 receptor further demonstrated its requirement in the expression of sex determining region Y-box 18 (sox18) and nuclear receptor subfamily 2, group F, member 2 (nr2f2), essential factors of the lymphatic specification. The EP3 receptor was expressed in the posterior cardinal vein (region of embryonic lymphatic development) and the adjacent intermediate cell mass (ICM) during the lymphatic specification. COX1 was expressed in the region more upstream of the posterior cardinal vein relative to the EP3 receptor, and the COX1-selective inhibitor impaired the lymphatic specification. On the other hand, two COX2 subtypes did not show distinct sites of expression around the region of expression of the EP3 receptor. Finally, we generated EP3-deficient zebrafish, which also showed defect in lymphatic specification and development. Thus, we demonstrated that COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of key factors for the lymphatic specification.

Characteristics of Multi-Organ Lymphangiectasia Resulting from Temporal Deletion of Calcitonin Receptor-Like Receptor in Adult Mice

Adrenomedullin (AM) and its receptor complexes, calcitonin receptor-like receptor (Calcrl) and receptor activity modifying protein 2/3, are highly expressed in lymphatic endothelial cells and are required for embryonic lymphatic development. To determine the role of Calcrl in adulthood, we used an inducible Cre-loxP system to temporally and ubiquitously delete Calcrl in adult mice. Following tamoxifen injection, Calcrlfl/fl/CAGGCre-ER™ mice rapidly developed corneal edema and inflammation that was preceded by and persistently associated with dilated corneoscleral lymphatics. Lacteals and submucosal lymphatic capillaries of the intestine were also dilated, while mesenteric collecting lymphatics failed to properly transport chyle after an acute Western Diet, culminating in chronic failure of Calcrlfl/fl/CAGGCre-ER™ mice to gain weight. Dermal lymphatic capillaries were also dilated and chronic edema challenge confirmed significant and prolonged dermal lymphatic insufficiency. In vivo and in vitro imaging of lymphatics with either genetic or pharmacologic inhibition of AM signaling revealed markedly disorganized lymphatic junctional proteins ZO-1 and VE-cadherin. The maintenance of AM signaling during adulthood is required for preserving normal lymphatic permeability and function. Collectively, these studies reveal a spectrum of lymphatic defects in adult Calcrlfl/fl/CAGGCre-ER™ mice that closely recapitulate the clinical symptoms of patients with corneal, intestinal and peripheral lymphangiectasia.

Zebrafish prox1b Mutants Develop a Lymphatic Vasculature, and prox1b Does Not Specifically Mark Lymphatic Endothelial Cells

BackgroundThe expression of the Prospero homeodomain transcription factor (Prox1) in a subset of cardinal venous cells specifies the lymphatic lineage in mice. Prox1 is also indispensible for the maintenance of lymphatic cell fate, and is therefore considered a master control gene for lymphangiogenesis in mammals. In zebrafish, there are two prox1 paralogues, the previously described prox1 (also known as prox1a) and the newly identified prox1b.Principal FindingsTo investigate the role of the prox1b gene in zebrafish lymphangiogenesis, we knocked-down prox1b and found that depletion of prox1b mRNA did not cause lymphatic defects. We also generated two different prox1b mutant alleles, and maternal-zygotic homozygous mutant embryos were viable and did not show any lymphatic defects. Furthermore, the expression of prox1b was not restricted to lymphatic vessels during zebrafish development.ConclusionWe conclude that Prox1b activity is not essential for embryonic lymphatic development in zebrafish.

Ets family members induce lymphangiogenesis through physical and functional interaction with Prox1

Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

COUP‐TFII regulates the functions of Prox1 in lymphatic endothelial cells through direct interaction

During embryonic lymphatic development, Prox1 homeobox transcription factor is expressed in a subset of venous blood vascular endothelial cells (BECs) in which COUP-TFII orphan nuclear receptor is highly expressed. Prox1 induces differentiation of BECs into lymphatic endothelial cells (LECs) by inducing the expression of various LEC markers including vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms of how transcriptional activities of Prox1 are regulated are largely unknown. In the present study, we show that COUP-TFII plays important roles in the regulation of the function of Prox1. In BECs and LECs, Prox1 promotes the proliferation and migration toward VEGF-C by inducing the expression of cyclin E1 and VEGFR3, respectively. Gain-of-function studies showed that COUP-TFII negatively regulates the effects of Prox1 in BECs and LECs whereas loss-of-function studies showed that COUP-TFII negatively and positively regulates Prox1 in BECs and LECs, respectively. We also show that endogenous Prox1 and COUP-TFII physically interact in LECs and that both Prox1 and COUP-TFII bind to the endogenous cyclin E1 promoter. These results suggest that COUP-TFII physically and functionally interact during differentiation and maintenance of lymphatic vessels.

A hypothesis on the role of primitive macrophages in initial embryonic lymphatic development

Recent evidences have shown that macrophages are tightly related to pathological lymphangiogenesis. However, the effects which primitive macrophages take in embryonic lymphatic development remains unclear. Here, we postulate that the primitive macrophages may play an important role in initial embryonic lymphatic development. The possible mechanism: primitive macrophages induce BECs to transdifferentiate into LECs and initiate the budding, moreover, the lymph sacs are not only formed by LECs but also some scattered cells with macrophage characteristics preferentially located in the loose mesenchyme.

Role of VEGF-D and VEGFR-3 in developmental lymphangiogenesis, a chemicogenetic study in Xenopus tadpoles

AbstractThe importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

Prox1 Induces Lymphatic Endothelial Differentiation via Integrin α9 and Other Signaling Cascades

During embryonic lymphatic development, a homeobox transcription factor Prox1 plays important roles in sprouting and migration of a subpopulation of blood vessel endothelial cells (BECs) toward VEGF-C-expressing cells. However, effects of Prox1 on endothelial cellular behavior remain to be elucidated. Here, we show that Prox1, via induction of integrin alpha9 expression, inhibits sheet formation and stimulates motility of endothelial cells. Prox1-expressing BECs preferentially migrated toward VEGF-C via up-regulation of the expression of integrin alpha9 and VEGF receptor 3 (VEGFR3). In mouse embryos, expression of VEGFR3 and integrin alpha9 is increased in Prox1-expressing lymphatic endothelial cells (LECs) compared with BECs. Knockdown of Prox1 expression in human LECs led to decrease in the expression of integrin alpha9 and VEGFR3, resulting in the decreased chemotaxes toward VEGF-C. These findings suggest that Prox1 plays important roles in conferring and maintaining the characteristics of LECs by modulating multiple signaling cascades and that integrin alpha9 may function as a key regulator of lymphangiogenesis acting downstream of Prox1.

1135: Development of a Three-Dimensional Anisotropic Pelvic Lymph Node Clinical Target Volume and Planning Target Volume Using Magnetic Resonance Lymphadenography With Ferumoxtran-10

1135: Development of a Three-Dimensional Anisotropic Pelvic Lymph Node Clinical Target Volume and Planning Target Volume Using Magnetic Resonance Lymphadenography With Ferumoxtran-10

Embryonic Lymphatic Development: Recent Advances and Unanswered Questions

Lymphatic Research and BiologyVol. 3, No. 3 Original PapersEmbryonic Lymphatic Development: Recent Advances and Unanswered QuestionsNatasha L. HarveyNatasha L. HarveySearch for more papers by this authorPublished Online:28 Sep 2005https://doi.org/10.1089/lrb.2005.3.157AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byFunctions and Regeneration of Mature Cardiac Lymphatic Vessels in Atherosclerosis, Myocardial Infarction, and Heart Failure Yaqi Zhang, Yingnan Bai, Qing Jing, and Juying Qian13 December 2018 | Lymphatic Research and Biology, Vol. 16, No. 6Early life events influence whole-of-life metabolic health via gut microflora and gut permeability19 March 2014 | Critical Reviews in Microbiology, Vol. 41, No. 3Classification of Lymphatic-system Malformations in Primary Lymphoedema based on MR LymphangiographyEuropean Journal of Vascular and Endovascular Surgery, Vol. 44, No. 3 Volume 3Issue 3Sep 2005 InformationCopyright 2005, Mary Ann Liebert, Inc.To cite this article:Natasha L. Harvey.Embryonic Lymphatic Development: Recent Advances and Unanswered Questions.Lymphatic Research and Biology.Sep 2005.157-165.http://doi.org/10.1089/lrb.2005.3.157Published in Volume: 3 Issue 3: September 28, 2005PDF download