Expression of lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) in the human placenta.
ABSTRACT: Lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) is a selective marker for lymphatic endothelium and a homolog of CD44, the hyaluronan (HA) receptor. HA in the extracellular matrix plays roles in tissue remodeling, development, and homeostasis, and as an HA receptor, LYVE-1 mediated HA metabolism might regulate these events. Currently, little is known about the lymphatic character within the human placenta. This study therefore determined LYVE-1 and other lymphatic markers in human placentas.Placentas and villous tissue were fixed and immunostained for human LYVE-1 and CD44 and examined by RT-PCR. LYVE-1 was expressed at both protein and mRNA levels in trophoblast cells (TC) and in villous core endothelium (VCE). Predominant protein expression for LYVE-1 was observed in syncytiotrophoblast cells (TCs) of preterm placentas. Neither mRNA or protein for CD44 was expressed. Other blood and lymphatic-lineage molecules (VEGF-A, -C, and -D, Flt-1, KDR, Flt-4, and Prox-1) were examined by RT-PCR. VEGF-A, VEGF-D, and Flt-1 mRNA were observed in TCs and VCEs, while mRNA for VEGF-C, KDR, and Flt-4 was mainly observed in VCEs. Prox-1 was found at the mRNA, but not protein level in TCs and VCEs. Our findings indicate (1) the importance of LYVE-1, but not CD44, in regulation of HA metabolism in the maternal-fetal interface and fetal circulation, and (2) possible dual blood and lymphatic phenotypic characteristics in fetal endothelium. These results provide new insights into HA metabolism and lymphatic-lineage molecule expression in the human placenta.
Project description:The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just before septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium, which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart.
Project description:The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.
Project description:The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.
Project description:Hyaluronan (HA), a large nonsulfated glycosaminogycan in the extracellular matrix, whose degraded fragments termed as low molecular weight hyaluronan (LMW-HA), has been reported as an important regulator of angiogenesis. However, little is known about the influence of LMW-HA on lymphangiogenesis. In this study, we try to explore the in vitro effects of LMW-HA on lymphangiogenesis and identify the underlying molecular mechanisms. Our results showed that LMW-HA stimulation significantly increased lymphatic endothelial cells (LECs) proliferation, migration and tube formation. Further experiments demonstrated that LMW-HA altered actin cytoskeleton rearrangement and increased the formation of intense stress fibers, lamellipodia and filopodia. Mechanistically, LMW-HA stimulation resulted in rapid tyrosine phosphorylation of protein kinase C ?/?II (PKC?/?II) and extracellular-regulated kinase 1/2 (ERK1/2). Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a homologue of CD44, is the main cell surface receptor for HA in LECs. Blocking the binding interaction of LMW-HA with LYVE-1 using neutralizing anti-LYVE-1 antibodies significantly inhibited LECs proliferation, migration, tube formation and signal transduction induced by LMW-HA, suggesting that LMW-HA may play a critical role in the processes required for lymphangiogenesis through interactions with its receptor LYVE-1 and triggering intracellular signal cascades.
Project description:Lymphatic vessels play a key role in maintaining tissue-fluid homeostasis, immune surveillance and metastasis. The hyaluronan receptor, LYVE-1, is widely used as a molecular marker for adult and embryonic lymphatic endothelium, but its physiological functions have not yet been established in vivo. In agreement with a recent report, LYVE-1(-/-) mice, which are healthy and fertile, do not display any defects related to congenital abnormalities of the lymphatic system. One hypothesis for the absence of a phenotype in LYVE-1 null mice is that other hyaluronan receptors, such as CD44, may compensate for LYVE-1. To test this hypothesis, we created LYVE-1/CD44 double knockout mice with appropriate littermate controls. Lymphatic vessel structure and function, as determined by histological methods and intravital microscopy, show that LYVE-1(-/-), CD44(-/-) and LYVE-1(-/-)/CD44(-/-) mice are indistinguishable from wild-type mice under normal conditions. Furthermore, resolution of carrageenan-induced paw edema is comparable in all genotypes. However, LYVE-1(-/-)/CD44(-/-) mice exhibit increased edema formation in a carrageenan-induced paw inflammation model compared to wild-type mice, but not to LYVE(-/-) or CD44(-/-) mice. These data suggest that LYVE-1 and CD44 are not required for the formation or function of lymphatics, but do not rule out a role for LYVE-1 in inflammation.
Project description:CRSBP-1 (mammalian LYVE-1) is a membrane glycoprotein highly expressed in lymphatic endothelial cells (LECs). It has multiple ligands, including hyaluronic acid (HA) and growth factors/cytokines (e.g., PDGF-BB and VEGF-A) containing CRS motifs (clusters of basic amino-acid residues). The ligand binding activities are mediated by Link module and acidic-amino-acid-rich (AAAR) domains, respectively. These CRSBP-1/LYVE-1 ligands have been shown to induce opening of lymphatic intercellular junctions in LEC monolayers and in lymphatic vessels in wild-type mice. We hypothesize that CRSBP-1/LYVE-1 ligands, particularly CRS-containing growth factors/cytokines, are secreted by immune and cancer cells for lymphatic entry during adaptive immune responses and lymphatic metastasis. We have looked into the origin of the Link module and AAAR domain of LYVE-1 in evolution and its association with the development of lymph nodes and efficient adaptive immunity. Lymph nodes represent the only major recent innovation of the adaptive immune systems in evolution particularly to mammals and bird. Here we demonstrate that the development of the LYVE-1 gene with the AAAR domain in evolution is associated with acquisition of lymph nodes and adaptive immunity. LYVE-1 from other species, which have no lymph nodes, lack the AAAR domain and efficient adaptive immunity. Synthetic CRSBP-1 ligands PDGF and VEGF peptides, which contain the CRS motifs of PDGF-BB and VEGF-A, respectively, specifically bind to CRSBP-1 but do not interact with either PDGFβR or VEGFR2. These peptides function as adjuvants by enhancing adaptive immunity of pseudorabies virus (PRV) vaccine in pigs. These results support the notion that LYVE-1 is involved in adaptive immunity in mammals.
Project description:Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFR?, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFR? or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development.
Project description:Although bronchial angiogenesis has been well documented in allergic asthma, lymphangiogenesis has not been widely studied. Therefore, we evaluated changes in lung lymphatics in a rat model of allergen-induced asthma using house dust mite (Der p 1; 100 ?g/challenge). Additionally, properties of isolated lung lymphatic endothelial cells (CD45-, CD141+, LYVE-1+, Prox-1+) were studied in vitro. Three weeks after the onset of intranasal allergen exposure (twice-weekly), an increase in the number of lung lymphatic vessels was measured (34% increase) by lung morphometry. New lymphatic structures were seen predominantly in the peribronchial and periarterial interstitial space but also surrounding large airways. Isolated lymphatic endothelial cells from sensitized lungs showed enhanced proliferation (% Ki67+), chemotaxis, and tube formation (number and length) compared to lymphatic endothelial cells isolated from naive rat lungs. This hyper-proliferative lymphangiogenic phenotype was preserved through multiple cell passages (2-8). Lymphatic endothelial cells isolated from naive and HDM-sensitized rats produced similar in vitro levels of VEGF-C, VEGF-D, and VEGFR3 protein, each recognized as critical lymphangiogenic factors. Inhibition with anti-VEGFR (axitinib, 0.1 ?M) blocked proliferation and chemotaxis. Results suggest that in vivo sensitization causes fundamental changes to lymphatic endothelium, which are retained in vitro, and may relate to VEGFR downstream signaling.
Project description:Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.
Project description:Schlemm's canal is an important structure of the conventional aqueous humor outflow pathway and is critically involved in regulating the intraocular pressure. In this study, we report a novel finding that prospero homeobox protein 1 (Prox-1), the master control gene for lymphatic development, is expressed in Schlemm's canal. Moreover, we provide a novel in vivo method of visualizing Schlemm's canal using a transgenic mouse model of Prox-1-green fluorescent protein (GFP). The anatomical location of Prox-1? Schlemm's canal was further confirmed by in vivo gonioscopic examination and ex vivo immunohistochemical analysis. Additionally, we show that the Schlemm's canal is distinguishable from typical lymphatic vessels by lack of lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expression and absence of apparent sprouting reaction when inflammatory lymphangiogenesis occurred in the cornea. Taken together, our findings offer new insights into Schlemm's canal and provide a new experimental model for live imaging of this critical structure to help further our understanding of the aqueous humor outflow. This may lead to new avenues toward the development of novel therapeutic intervention for relevant diseases, most notably glaucoma.