Ephrin-B2 expression critically influences Nipah virus infection independent of its cytoplasmic tail

<p>Abstract</p> <p>Background</p> <p>Cell entry and cell-to-cell spread of the highly pathogenic Nipah virus (NiV) requires binding of the NiV G protein to cellular ephrin receptors and subsequent NiV F-mediated fusion. Since expression levels of the main NiV entry receptor ephrin-B2 (EB2) are highly regulated <it>in vivo </it>to fulfill the physiological functions in axon guidance and angiogenesis, the goal of this study was to determine if changes in the EB2 expression influence NiV infection.</p> <p>Results</p> <p>Surprisingly, transfection of increasing EB2 plasmid concentrations reduced cell-to-cell fusion both in cells expressing the NiV glycoproteins and in cells infected with NiV. This effect was attributed to the downregulation of the NiV glycoproteins from the cell surface. In addition to the influence on cell-to-cell fusion, increased EB2 expression significantly reduced the total amount of NiV-infected cells, thus interfered with virus entry. To determine if the negative effect of elevated EB2 expression on virus entry is a result of an increased EB2 signaling, receptor function of a tail-truncated and therefore signaling-defective ΔcEB2 was tested. Interestingly, ΔcEB2 fully functioned as NiV entry and fusion receptor, and overexpression also interfered with virus replication.</p> <p>Conclusion</p> <p>Our findings clearly show that EB2 signaling does not account for the striking negative impact of elevated receptor expression on NiV infection, but rather that the ratio between the NiV envelope glycoproteins and surface receptors critically influence cell-to-cell fusion and virus entry.</p

Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2

The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly induced by COMP-Ang1 also in transfected cells that do not express Tie2. When cotransfected, Tie2 formed heteromeric complexes with Tie1, enhanced Tie1 activation, and induced phosphorylation of a kinase-inactive Tie1 in a ligand-dependent manner. Tie1 phosphorylation was also induced by native Ang1 and Ang4, although less efficiently than with COMP-Ang1. In conclusion, we show that Tie1 phosphorylation is induced by multiple angiopoietin proteins and that the activation is amplified via Tie2. These results should be important in dissecting the signal transduction pathways and biological functions of Tie1

Unique Cell Type-Specific Junctional Complexes in Vascular Endothelium of Human and Rat Liver Sinusoids

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis

Visceral obesity and insulin resistance associate with CD36 deletion in lymphatic endothelial cells

Disruption of lymphatic lipid transport is linked to obesity and type 2 diabetes (T2D), but regulation of lymphatic vessel function and its link to disease remain unclear. Here we show that intestinal lymphatic endothelial cells (LECs) have an increasing CD36 expression from lymphatic capillaries (lacteals) to collecting vessels, and that LEC CD36 regulates lymphatic integrity and optimizes lipid transport. Inducible deletion of CD36 in LECs in adult mice (Cd36(ΔLEC)) increases discontinuity of LEC VE-cadherin junctions in lacteals and collecting vessels. Cd36(ΔLEC) mice display slower transport of absorbed lipid, more permeable mesenteric lymphatics, accumulation of inflamed visceral fat and impaired glucose disposal. CD36 silencing in cultured LECs suppresses cell respiration, reduces VEGF-C-mediated VEGFR2/AKT phosphorylation and destabilizes VE-cadherin junctions. Thus, LEC CD36 optimizes lymphatic junctions and integrity of lymphatic lipid transport, and its loss in mice causes lymph leakage, visceral adiposity and glucose intolerance, phenotypes that increase risk of T2D

A novel SEMA3G mutation in two siblings affected by syndromic GnRH deficiency

Introduction: Gonadotropin-releasing hormone (GnRH) deficiency causes hypogonadotropic hypogonadism (HH), a rare genetic disorder that impairs sexual reproduction. HH can be due to defective GnRH-secreting neuron development or function and may be associated with other clinical signs in overlapping genetic syndromes. With most of the cases being idiopathic, genetics underlying HH is still largely unknown. Objective: To assess the contribution of mutated Semaphorin 3G (SEMA3G) gene in the onset of a syndromic form of HH, characterized by intellectual disabilities and facial dysmorphic features. Method: By combining homozygosity mapping with exome sequencing, we identified a novel variant in SEMA3G gene. We then applied mouse as a model organism to examine SEMA3G expression and its functional requirement in vivo. Further, we applied homology modelling in silico and cell culture assays in vitro to validate the pathogenicity of the identified gene variant. Results: We found that: SEMA3G is expressed along the migratory route of GnRH neurons and in the developing pituitary; SEMA3G affects GnRH neuron development, but is redundant in the adult hypothalamic-pituitary-gonadal axis; mutated SEMA3G alters binding properties in silico and in vitro to its PlexinAs receptors and attenuates its effect on the migration of immortalized GnRH neurons. Conclusion: In silico, in vitro and in vivo models revealed that SEMA3G regulates GnRH neuron migration and that its mutation affecting receptor selectivity may be responsible for the HH-related defects

Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing

Background Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively. Methods We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC). Results The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone. Conclusions The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Publisher Copyright: © 2022 American Society for Clinical Investigation. All rights reserved.Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with smallmolecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.Peer reviewe

Primary tumor–derived systemic nANGPTL4 inhibits metastasis

Primary tumors and distant site metastases form a bidirectionally communicating system. Yet, the molecular mechanisms of this crosstalk are poorly understood. Here, we identified the proteolytically cleaved fragments of angiopoietin-like 4 (ANGPTL4) as contextually active protumorigenic and antitumorigenic contributors in this communication ecosystem. Preclinical studies in multiple tumor models revealed that the C-terminal fragment (cANGPTL4) promoted tumor growth and metastasis. In contrast, the N-terminal fragment of ANGPTL4 (nANGPTL4) inhibited metastasis and enhanced overall survival in a postsurgical metastasis model by inhibiting WNT signaling and reducing vascularity at the metastatic site. Tracing ANGPTL4 and its fragments in tumor patients detected full-length ANGPTL4 primarily in tumor tissues, whereas nANGPTL4 predominated in systemic circulation and correlated inversely with disease progression. The study highlights the spatial context of the proteolytic cleavage-dependent pro- and antitumorigenic functions of ANGPTL4 and identifies and validates nANGPTL4 as a novel biomarker of tumor progression and antimetastatic therapeutic agent